Dual variable domain immunoglobulin and uses thereof

ABSTRACT

The present invention relates to engineered multivalent and multispecific binding proteins, methods of making, and specifically to their uses in the prevention and/or treatment of acute and chronic inflammatory and other diseases.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional application of U.S. Serial No.11/507,050, filed Aug. 18, 2006, now U.S. Pat. No. 7,612,181, whichclaims the benefit of priority to U.S. Provisional Application No.60/709,911, filed Aug. 19, 2005, and U.S. Provisional Application No.60/732,892, filed Nov. 2, 2005.

FIELD OF THE INVENTION

The present invention relates to multivalent and multispecific bindingproteins, methods of making, and specifically to their uses in theprevention and/or treatment of acute and chronic inflammatory, cancer,and other diseases.

BACKGROUND OF THE INVENTION

Engineered proteins, such as multispecific antibodies capable of bindingtwo or more antigens are known in the art. Such multispecific bindingproteins can be generated using cell fusion, chemical conjugation, orrecombinant DNA techniques.

Bispecific antibodies have been produced using the quadroma technology(see Milstein, C. and A. C. Cuello, Nature, 1983. 305(5934): p. 537-40)based on the somatic fusion of two different hybridoma cell linesexpressing murine monoclonal antibodies with the desired specificitiesof the bispecific antibody. Because of the random pairing of twodifferent Ig heavy and light chains within the resultinghybrid-hybridoma (or quadroma) cell line, up to ten differentimmunogloblin species are generated of which only one is the functionalbispecific antibody. The presence of mispaired by-products, andsignificantly reduced production yields, means sophisticatedpurification procedures are required.

Bispecific antibodies can be produced by chemical conjugation of twodifferent mAbs (see Staerz, U. D., et al., Nature, 1985. 314(6012): p.628-31). This approach does not yield homogeneous preparation. Otherapproaches have used chemical conjugation of two different monoclonalantibodies or smaller antibody fragments (see Brennan, M., et al.,Science, 1985. 229(4708): p. 81-3). Another method is the coupling oftwo parental antibodies with a hetero-bifunctional crosslinker, but theresulting preparations of bispecific antibodies suffer from significantmolecular heterogeneity because reaction of the crosslinker with theparental antibodies is not site-directed. To obtain more homogeneouspreparations of bispecific antibodies two different Fab fragments havebeen chemically crosslinked at their hinge cysteine residues in asite-directed manner (see Glennie, M. J., et al., J Immunol, 1987.139(7): p. 2367-75). But this method results in Fab′2 fragments, notfull IgG molecule.

A wide variety of other recombinant bispecific antibody formats havebeen developed in the recent past (see Kriangkum, J., et al., BiomolEng, 2001. 18(2): p. 31-40). Amongst them tandem single-chain Fvmolecules and diabodies, and various derivatives there of, are the mostwidely used formats for the construction of recombinant bispecificantibodies. Routinely, construction of these molecules starts from twosingle-chain Fv (scFv) fragments that recognize different antigens (seeEconomides, A. N., et al., Nat Med, 2003. 9(1): p. 47-52). Tandem scFvmolecules (taFv) represent a straightforward format simply connectingthe two scFv molecules with an additional peptide linker. The two scFvfragments present in these tandem scFv molecules form separate foldingentities. Various linkers can be used to connect the two scFv fragmentsand linkers with a length of up to 63 residues (see Nakanishi, K., etal., Annu Rev Immunol, 2001. 19: p. 423-74). Although the parental scFvfragments can normally be expressed in soluble form in bacteria, it is,however, often observed that tandem scFv molecules form insolubleaggregates in bacteria. Hence, refolding protocols or the use ofmammalian expression systems are routinely applied to produce solubletandem scFv molecules. In a recent study, in vivo expression bytransgenic rabbits and cattle of a tandem scFv directed against CD28 anda melanoma-associated proteoglycan was reported (see Gracie, J. A., etal., J Clin Invest, 1999. 104(10): p. 1393-401). In this construct, thetwo scFv molecules were connected by a CH1 linker and serumconcentrations of up to 100 mg/L of the bispecific antibody were found.Various strategies including variations of the domain order or usingmiddle linkers with varying length or flexibility were employed to allowsoluble expression in bacteria. A few studies have now reportedexpression of soluble tandem scFv molecules in bacteria (see Leung, B.P., et al., J Immunol, 2000. 164(12): p. 6495-502; Ito, A., et al., JImmunol, 2003. 170(9): p. 4802-9; Karni, A., et al., J Neuroimmunol,2002. 125(1-2): p. 134-40) using either a very short Ala3 linker or longglycine/serine-rich linkers. In a recent study, phage display of atandem scFv repertoire containing randomized middle linkers with alength of 3 or 6 residues was employed to enrich for those moleculesthat are produced in soluble and active form in bacteria. This approachresulted in the isolation of a preferred tandem scFv molecule with a 6amino acid residue linker (see Arndt, M. and J. Krauss, Methods MolBiol, 2003. 207: p. 305-21). It is unclear whether this linker sequencerepresents a general solution to the soluble expression of tandem scFvmolecules. Nevertheless, this study demonstrated that phage display oftandem scFv molecules in combination with directed mutagenesis is apowerful tool to enrich for these molecules, which can be expressed inbacteria in an active form.

Bispecific diabodies (Db) utilize the diabody format for expression.Diabodies are produced from scFv fragments by reducing the length of thelinker connecting the VH and VL domain to approximately 5 residues (seePeipp, M. and T. Valerius, Biochem Soc Trans, 2002. 30(4): p. 507-11).This reduction of linker size facilitates dimerization of twopolypeptide chains by crossover pairing of the VH and VL domains.Bispecific diabodies are produced by expressing, two polypeptide chainswith, either the structure VHA-VLB and VHB-VLA (VH-VL configuration), orVLA-VHB and VLB-VHA (VL-VH configuration) within the same cell. A largevariety of different bispecific diabodies have been produced in the pastand most of them cab be expressed in soluble form in bacteria. However,a recent comparative study demonstrates that the orientation of thevariable domains can influence expression and formation of activebinding sites (see Mack, M., G. Riethmuller, and P. Kufer, Proc NatlAcad Sci USA, 1995. 92(15): p. 7021-5). Nevertheless, soluble expressionin bacteria represents an important advantage over tandem scFvmolecules. However, since two different polypeptide chains are expressedwithin a single cell inactive homodimers can be produced together withactive heterodimers. This necessitates the implementation of additionalpurification steps in order to obtain homogenous preparations ofbispecific diabodies. One approach to force the generation of bispecificdiabodies is the production of knob-into-hole diabodies (see Holliger,P., T. Prospero, and G. Winter, Proc Natl Acad Sci USA, 1993. 90(14): p.6444-8.18). This was demonstrated for a bispecific diabody directedagainst HER2 and CD3. A large knob was introduced in the VH domain byexchanging Val37 with Phe and Leu45 with Trp and a complementary holewas produced in the VL domain by mutating Phe98 to Met and Tyr87 to Ala,either in the anti-HER2 or the anti-CD3 variable domains. By using thisapproach the production of bispecific diabodies could be increased from72% by the parental diabody to over 90% by the knob-into-hole diabody.Importantly, production yields did only slightly decrease as a result ofthese mutations. However, a reduction in antigen-binding activity wasobserved for several analyzed constructs. Thus, this rather elaborateapproach requires the analysis of various constructs in order toidentify those mutations that produce heterodimeric molecule withunaltered binding activity. In addition, such approach requiresmutational modification of the immunoglobulin sequence at the constantregion, thus creating non-native and non-natural form of the antibodysequence, which may result in increased immunogenicity, poor in vivostability, as well as undesirable pharmacokinetics.

Single-chain diabodies (scDb) represent an alternative strategy toimprove the formation of bispecific diabody-like molecules (seeHolliger, P. and G. Winter, Cancer Immunol Immunother, 1997. 45(3-4): p.128-30; Wu, A. M., et al., Immunotechnology, 1996. 2(1): p. 21-36).Bispecific single-chain diabodies are produced by connecting the twodiabody-forming polypeptide chains with an additional middle linker witha length of approximately 15 amino acid residues. Consequently, allmolecules with a molecular weight corresponding to monomericsingle-chain diabodies (50-60 kDa) are bispecific. Several studies havedemonstrated that bispecific single chain diabodies are expressed inbacteria in soluble and active form with the majority of purifiedmolecules present as monomers (see Holliger, P. and G. Winter, CancerImmunol Immunother, 1997. 45(3-4): p. 128-30; Wu, A. M., et al.,Immunotechnology, 1996. 2(1): p. 21-36; Pluckthun, A. and P. Pack,Immunotechnology, 1997. 3(2): p. 83-105; Ridgway, J. B., et al., ProteinEng, 1996. 9(7): p. 617-21). Thus, single-chain diabodies combine theadvantages of tandem scFvs (all monomers are bispecific) and diabodies(soluble expression in bacteria).

More recently diabody have been fused to Fc to generate more Ig-likemolecules, named di-diabody (see Lu, D., et al., J Biol Chem, 2004.279(4): p. 2856-65). In addition, multivalent antibody constructcomprising two Fab repeats in the heavy chain of an IgG and capable ofbinding four antigen molecules has been described (see WO 0177342A1, andMiller, K., et al., J Immunol, 2003. 170(9): p. 4854-61).

There is a need in the art for improved multivalent binding proteinscapable of binding two or more antigens. The present invention providesa novel family of binding proteins capable of binding two or moreantigens with high affinity.

SUMMARY OF THE INVENTION

This invention pertains to multivalent binding proteins capable ofbinding two or more antigens. The present invention provides a novelfamily of binding proteins capable of binding two or more antigens withhigh affinity.

In one embodiment the invention provides a binding protein comprising apolypeptide chain, wherein said polypeptide chain comprisesVD1-(X1)n-VD2-C—(X2)n, wherein VD1 is a first variable domain, VD2 is asecond variable domain, C is a constant domain, X1 represents an aminoacid or polypeptide, X2 represents an Fc region and n is 0 or 1. In apreferred embodiment the VD1 and VD2 in the binding protein are heavychain variable domains. More preferably the heavy chain variable domainis selected from the group consisting of a murine heavy chain variabledomain, a human heavy chain variable domain, a CDR grafted heavy chainvariable domain, and a humanized heavy chain variable domain. In apreferred embodiment VD1 and VD2 are capable of binding the sameantigen. In another embodiment VD1 and VD2 are capable of bindingdifferent antigens. Preferably C is a heavy chain constant domain. Morepreferably X1 is a linker with the proviso that X1 is not CH1. Mostpreferably X1 is a linker selected from the group consisting ofAKTTPKLEEGEFSEAR (SEQ ID NO: 118); AKTTPKLEEGEFSEARV (SEQ ID NO: 119);AKTTPKLGG (SEQ ID NO:120); SAKTTPKLGG (SEQ ID NO:121); SAKTTP (SEQ IDNO:122); RADAAP (SEQ ID NO: 123); RADAAPTVS (SEQ ID NO: 124);RADAAAAGGPGS (SEQ ID NO: 125); RADAAAA(G₄S)₄ (SEQ ID NO: 126);SAKTTPKLEEGEFSEARV (SEQ ID NO: 127); ADAAP (SEQ ID NO:40); ADAAPTVSIFPP(SEQ ID NO: 103); TVAAP (SEQ ID NO:44); TVAAPSVFIFPP (SEQ ID NO:50);QPKAAP (SEQ ID NO:88); QPKAAPSVTLFPP (SEQ ID NO:92); AKTTPP (SEQ IDNO:38); AKTTPPSVTPLAP (SEQ ID NO: 128); AKTTAP (SEQ ID NO: 129);AKTTAPSVYPLAP (SEQ ID NO:99); ASTKGP (SEQ ID NO:42); ASTKGPSVFPLAP (SEQID NO:48), GGGGSGGGGSGGGGS (SEQ ID NO: 130); GENKVEYAPALMALS (SEQ ID NO:131); GPAKELTPLKEAKVS (SEQ ID NO: 132); and GHEAAAVMQVQYPAS (SEQ IDNO:133). Preferably X2 is an Fc region. More preferably X2 is a variantFc region.

In a preferred embodiment the binding protein disclosed above comprisesa polypeptide chain, wherein said polypeptide chain comprisesVD1-(X1)n-VD2-C—(X2)n, wherein VD1 is a first heavy chain variabledomain, VD2 is a second heavy chain variable domain, C is a heavy chainconstant domain, X1 is a linker with the proviso that it is not CH1, andX2 is an Fc region.

In another embodiment VD1 and VD2 in the binding protein are light chainvariable domains. Preferably the light chain variable domain is selectedfrom the group consisting of a murine light chain variable domain, ahuman light chain variable domain, a CDR grafted light chain variabledomain, and a humanized light chain variable domain. In one embodimentVD1 and VD2 are capable of binding the same antigen. In anotherembodiment VD1 and VD2 are capable of binding different antigens.Preferably C is a light chain constant domain. More preferably X1 is alinker with the proviso that X1 is not CL1. Preferably X1 is a linkerselected from the group consisting of AKTTPKLEEGEFSEAR (SEQ ID NO: 118);AKTTPKLEEGEFSEARV (SEQ ID NO: 119); AKTTPKLGG (SEQ ID NO: 120);SAKTTPKLGG (SEQ ID NO: 121); SAKTTP (SEQ ID NO: 122); RADAAP (SEQ ID NO:123); RADAAPTVS (SEQ ID NO: 124); RADAAAAGGPGS (SEQ ID NO: 125);RADAAAA(G₄S)₄ (SEQ ID NO: 126); SAKTTPKLEEGEFSEARV (SEQ ID NO: 127);ADAAP (SEQ ID NO:40); ADAAPTVSIFPP (SEQ ID NO: 103); TVAAP (SEQ IDNO:44); TVAAPSVFIFPP (SEQ ID NO:50); QPKAAP (SEQ ID NO:88);QPKAAPSVTLFPP (SEQ ID NO:92); AKTTPP (SEQ ID NO:38); AKTTPPSVTPLAP (SEQID NO: 128); AKTTAP (SEQ ID NO: 129); AKTTAPSVYPLAP (SEQ ID NO:99);ASTKGP (SEQ ID NO:42); and ASTKGPSVFPLAP (SEQ ID NO:48). Preferably thebinding protein does not comprise X2.

In a preferred embodiment the binding protein disclosed above comprisesa polypeptide chain, wherein said polypeptide chain comprisesVD1-(X1)n-VD2-C—(X2)n, wherein VD1 is a first light chain variabledomain, VD2 is a second light chain variable domain, C is a light chainconstant domain, X1 is a linker with the proviso that it is not CH1, andX2 does not comprise an Fc region.

In another preferred embodiment the invention provides a binding proteincomprising two polypeptide chains, wherein said first polypeptide chaincomprises VD1-(X1)n-VD2-C—(X2)n, wherein VD1 is a first heavy chainvariable domain, VD2 is a second heavy chain variable domain, C is aheavy chain constant domain, X1 is a linker with the proviso that it isnot CH1, and X2 is an Fc region; and said second polypeptide chaincomprises VD1-(X1)n-VD2-C—(X2)n, wherein VD1 is a first light chainvariable domain, VD2 is a second light chain variable domain, C is alight chain constant domain, X1 is a linker with the proviso that it isnot CH1, and X2 does not comprise an Fc region. Most preferably the DualVariable Domain (DVD) binding protein comprises four polypeptide chainswherein the first two polypeptide chains comprisesVD1-(X1)n-VD2-C—(X2)n, respectively wherein VD1 is a first heavy chainvariable domain, VD2 is a second heavy chain variable domain, C is aheavy chain constant domain, X1 is a linker with the proviso that it isnot CH1, and X2 is an Fc region; and the second two polypeptide chaincomprises VD1-(X1)n-VD2-C—(X2)n respectively, wherein VD1 is a firstlight chain variable domain, VD2 is a second light chain variabledomain, C is a light chain constant domain, X1 is a linker with theproviso that it is not CH1, and X2 does not comprise an Fc region. Sucha Dual Variable Domain (DVD) protein has four antigen binding sites.

In another preferred embodiment the binding proteins disclosed above arecapable of binding one or more targets. Preferably the target isselected from the group consisting of cytokines, cell surface proteins,enzymes and receptors. Preferably the binding protein is capable ofmodulating a biological function of one or more targets. More preferablythe binding protein is capable of neutralizing one or more targets. Thebinding protein of the invention is capable of binding cytokinesselected from the group consisting of lymphokines, monokines, andpolypeptide hormones. In a specific embodiment the binding protein iscapable of binding pairs of cytokines selected from the group consistingof IL-1α and IL-1β; IL-12 and IL-18, TNFα and IL-23, TNFα and IL-13; TNFand IL-18; TNF and IL-12; TNF and IL-1beta; TNF and MIF; TNF and IL-17;and TNF and IL-15; TNF and VEGF; VEGFR and EGFR; IL-13 and IL-9; IL-13and IL-4; IL-13 and IL-5; IL-13 and IL-25; IL-13 and TARC; IL-13 andMDC; IL-13 and MIF; IL-13 and TGF-β; IL-13 and LHR agonist; IL-13 andCL25; IL-13 and SPRR2a; IL-13 and SPRR2b; IL-13 and ADAM8; and TNFα andPGE4, IL-13 and PED2, TNF and PEG2. In another embodiment the bindingprotein of the invention is capable of binding pairs of targets selectedfrom the group consisting of CD138 and CD20; CD138 and CD40; CD19 andCD20; CD20 and CD3; CD38 & CD138; CD38 and CD20; CD38 and CD40; CD40 andCD20; CD-8 and IL-6; CSPGs and RGM A; CTLA-4 and BTNO2; IGF1 and IGF2;IGF1/2 and Erb2B; IL-12 and TWEAK; IL-13 and IL-1β; MAG and RGM A; NgRand RGM A; NogoA and RGM A; OMGp and RGM A; PDL-1 and CTLA-4; RGM A andRGM B; Te38 and TNFα; TNFα and Blys; TNFα and CD-22; TNFα and CTLA-4;TNFα and GP130; TNFα and IL-12p40; and TNFα and RANK ligand.

In one embodiment, the binding protein capable of binding human IL-1αand human IL-1β comprises a DVD heavy chain amino acid sequence selectedfrom the group consisting of SEQ ID NO. 33, SEQ ID NO. 37, SEQ ID NO.41, SEQ ID NO. 45, SEQ ID NO. 47, SEQ ID NO. 51, SEQ ID NO. 53, SEQ IDNO. 55, SEQ ID NO. 57, and SEQ ID NO. 59; and a DVD light chain aminoacid sequence selected from the group consisting of SEQ ID NO. 35, SEQID NO. 39, SEQ ID NO. 43, SEQ ID NO. 46, SEQ ID NO. 49, SEQ ID NO. 52,SEQ ID NO. 54, SEQ ID NO. 56, SEQ ID NO. 58, and SEQ ID NO. 60. Inanother embodiment, the binding protein capable of binding murine IL-1αand murine IL-1β comprises a DVD heavy chain amino acid sequence SEQ IDNO. 105, and a DVD light chain amino acid sequence SEQ ID NO. 109.

In one embodiment, the binding protein capable of binding IL-12 andIL-18 comprises a DVD heavy chain amino acid sequence selected from thegroup consisting of SEQ ID NO. 83, SEQ ID NO. 90, SEQ ID NO. 93, SEQ IDNO. 95, and SEQ ID NO. 114; and a DVD light chain amino acid sequenceselected from the group consisting of SEQ ID NO. 86, SEQ ID NO. 91, SEQID NO. 94, SEQ ID NO. 46, SEQ ID NO. 96, and SEQ ID NO. 116.

In one embodiment the binding protein capable of binding CD20 and CD3comprises a DVD heavy chain amino acid sequence is SEQ ID NO. 97, and aDVD light chain SEQ ID NO. 101.

In another embodiment the binding protein of the invention is capable ofbinding one, two or more cytokines, cytokine-related proteins, andcytokine receptors selected from the group consisting of BMP1, BMP2,BMP3B (GDF10), BMP4, BMP6, BMP8, CSF1 (M-CSF), CSF2 (GM-CSF), CSF3(G-CSF), EPO, FGF1 (aFGF), FGF2 (bFGF), FGF3 (int-2), FGF4 (HST), FGF5,FGF6 (HST-2), FGF7 (KGF), FGF9, FGF10, FGF11, FGF12, FGF12B, FGF14,FGF16, FGF17, FGF19, FGF20, FGF21, FGF23, IGF1, IGF2, IFNA1, IFNA2,IFNA4, IFNA5, IFNA6, IFNA7, IFNB1, IFNG, IFNW1, FIL1, FIL1 (EPSILON),FIL1 (ZETA), IL1A, IL1B, IL2, IL3, IL4, IL5, IL6, IL7, IL8, IL9, IL10,IL11, IL12A, IL12B, IL13, IL14, IL15, IL16, IL17, IL17B, IL18, IL19,IL20, IL22, IL23, IL24, IL25, IL26, IL27, IL28A, IL28B, IL29, IL30,PDGFA, PDGFB, TGFA, TGFB1, TGFB2, TGFB3, LTA (TNF-b), LTB, TNF (TNF-a),TNFSF4 (OX40 ligand), TNFSF5 (CD40 ligand), TNFSF6 (FasL), TNFSF7 (CD27ligand), TNFSF8 (CD30 ligand), TNFSF9 (4-1BB ligand), TNFSF10 (TRAIL),TNFSF11 (TRANCE), TNFSF12 (APO3L), TNFSF13 (April), TNFSF13B, TNFSF14(HVEM-L), TNFSF15 (VEGI), TNFSF18, FIGF (VEGFD), VEGF, VEGFB, VEGFC,IL1R1, IL1R2, IL1RL1, IL1RL2, IL2RA, IL2RB, IL2RC, IL3RA, IL4R, IL5RA,IL6R, IL7R, IL8RA, IL8RB, IL9R, IL10RA, IL10RB, IL11RA, IL12RB1,IL12RB2, IL13RA1, IL13RA2, IL15RA, IL17R, IL18R1, IL20RA, IL21R, IL22R,IL1HY1, IL1RAP, IL1RAPL1, IL1RAPL2, IL1RN, IL6ST, IL18BP, IL18RAP,IL22RA2, AIF1, HGF, LEP (leptin), PTN, and THPO.

The binding protein of the invention is capable of binding one or morechemokines, chemokine receptors, and chemokine-related proteins selectedfrom the group consisting of CCL1 (I-309), CCL2 (MCP-1/MCAF), CCL3(MIP-1a), CCL4 (MIP-1b), CCL5 (RANTES), CCL7 (MCP-3), CCL8 (mcp-2),CCL11 (eotaxin), CCL13 (MCP-4), CCL15 (MIP-1d), CCL16 (HCC-4), CCL17(TARC), CCL18 (PARC), CCL19 (MIP-3b), CCL20 (MIP-3a), CCL21(SLC/exodus-2), CCL22 (MDC/STC-1), CCL23 (MPIF-1), CCL24(MPIF-2/eotaxin-2), CCL25 (TECK), CCL26 (eotaxin-3), CCL27 (CTACK/ILC),CCL28, CXCL1 (GRO1), CXCL2 (GRO2), CXCL3 (GRO3), CXCL5 (ENA-78), CXCL6(GCP-2), CXCL9 (MIG), CXCL10 (IP10), CXCL11 (I-TAC), CXCL12 (SDF1),CXCL13, CXCL14, CXCL16, PF4 (CXCL4), PPBP (CXCL7), CX3CL1 (SCYD1),SCYE1, XCL1 (lymphotactin), XCL2 (SCM-1b), BLR1 (MDR15), CCBP2(D6/JAB61), CCR1 (CKR1/HM145), CCR2 (mcp-1RB/RA), CCR3 (CKR3/CMKBR3),CCR4, CCR5 (CMKBR5/ChemR13), CCR6 (CMKBR6/CKR-L3/STRL22/DRY6), CCR7(CKR7/EBI1), CCR8 (CMKBR8/TER1/CKR-L1), CCR9 (GPR-9-6), CCRL1 (VSHK1),CCRL2 (L-CCR), XCR1 (GPR5/CCXCR1), CMKLR1, CMKOR1 (RDC1), CX3CR1 (V28),CXCR4, GPR2 (CCR10), GPR31, GPR81 (FKSG80), CXCR3 (GPR9/CKR-L2), CXCR6(TYMSTR/STRL33/Bonzo), HM74, IL8RA (IL8Ra), IL8RB (IL8Rb), LTB4R(GPR16), TCP10, CKLFSF2, CKLFSF3, CKLFSF4, CKLFSF5, CKLFSF6, CKLFSF7,CKLFSF8, BDNF, C5R1, CSF3, GRCC10 (C10), EPO, FY (DARC), GDF5, HIFIA,IL8, PRL, RGS3, RGS13, SDF2, SLIT2, TLR2, TLR4, TREM1, TREM2, and VHL.The binding protein of the invention is capable of binding cell surfaceprotein selected from the group consisting of integrins. The bindingprotein of the invention is capable of binding enzyme selected from thegroup consisting of kinases and proteases. The binding protein of theinvention is capable of binding receptor selected from the groupconsisting of lymphokine receptor, monokine receptor, and polypeptidehormone receptor.

In a preferred embodiment the binding protein is multivalent. Morepreferably the binding protein is multispecific. The multivalent and ormultispecific binding proteins described above have desirable propertiesparticularly from a therapeutic standpoint. For instance, themultivalent and or multispecific binding protein may (1) be internalized(and/or catabolized) faster than a bivalent antibody by a cellexpressing an antigen to which the antibodies bind; (2) be an agonistantibody; and/or (3) induce cell death and/or apoptosis of a cellexpressing an antigen which the multivalent antibody is capable ofbinding to. The “parent antibody” which provides at least one antigenbinding specificity of the multivalent and or multispecific bindingproteins may be one which is internalized (and/or catabolized) by a cellexpressing an antigen to which the antibody binds; and/or may be anagonist, cell death-inducing, and/or apoptosis-inducing antibody, andthe multivalent and or multispecific binding protein as described hereinmay display improvement(s) in one or more of these properties. Moreover,the parent antibody may lack any one or more of these properties, butmay be endowed with them when constructed as a multivalent bindingprotein as hereindescribed.

In another embodiment the binding protein of the invention has an onrate constant (Kon) to one or more targets selected from the groupconsisting of: at least about 10²M⁻¹ s⁻¹; at least about 10³M⁻¹ s⁻¹; atleast about 10⁴M⁻¹ s⁻¹; at least about 10⁵M⁻¹ s⁻¹; and at least about10⁶M⁻¹ s⁻¹, as measured by surface plasmon resonance. Preferably, thebinding protein of the invention has an on rate constant (Kon) to one ormore targets between 10²M⁻¹ s⁻¹ to 10³M⁻¹ s⁻¹; between 10³M⁻¹ s⁻¹ to10⁴M⁻¹ s⁻¹; between 10⁴M⁻¹ s⁻¹ to 10⁵M⁻¹ s⁻¹; or between 10⁵M⁻¹ s⁻¹ to10⁶M⁻¹ s⁻¹, as measured by surface plasmon resonance.

In another embodiment the binding protein has an off rate constant(Koff) for one or more targets selected from the group consisting of: atmost about 10⁻³ s⁻¹; at most about 10⁻⁴ s⁻¹; at most about 10⁻⁵ s⁻¹; andat most about 10⁻⁶ s⁻¹, as measured by surface plasmon resonance.Preferably, the binding protein of the invention has an off rateconstant (Koff) to one or more targets of 10⁻³ s⁻¹ to 10⁻⁴ s⁻¹; of 10⁻⁴s⁻¹ to 10⁻⁵ s⁻¹; or of 10⁻⁵ s⁻¹ to 10⁻⁶ s⁻¹, as measured by surfaceplasmon resonance.

In another embodiment the binding protein has a dissociation constant(K_(D)) to one or more targets selected from the group consisting of: atmost about 10⁻⁷ M; at most about 10⁻⁸ M; at most about 10⁻⁹ M; at mostabout 10⁻¹⁰ M; at most about 10⁻¹¹ M; at most about 10⁻¹² M; and at most10⁻¹³ M. Preferably, the binding protein of the invention has adissociation constant (K_(D)) to IL-12 or IL-23 of 10⁻⁷ M to 10⁻⁸ M; of10⁻⁸ M to 10⁻⁹ M; of 10⁻⁹ M to 10⁻¹⁰ M; of 10⁻¹⁰ to 10⁻¹¹ M; of 10⁻¹¹ Mto 10⁻¹² M; or of 10⁻¹² to M 10⁻¹³ M.

In another embodiment the binding protein described above is a conjugatefurther comprising an agent selected from the group consisting of; animmunoadhension molecule, an imaging agent, a therapeutic agent, and acytotoxic agent. Preferably the imaging agent is selected from the groupconsisting of a radiolabel, an enzyme, a fluorescent label, aluminescent label, a bioluminescent label, a magnetic label, and biotin.More preferably the imaging agent is a radiolabel selected from thegroup consisting of: ³H, ¹⁴C, ³⁵S, ⁹⁰ _(Y), ⁹⁹Tc, ¹¹¹In, ¹²⁵I, ¹³¹I,¹⁷⁷Lu, ¹⁶⁶Ho, and ¹⁵³Sm. Preferably the therapeutic or cytotoxic agentis selected from the group consisting of; an anti-metabolite, analkylating agent, an antibiotic, a growth factor, a cytokine, ananti-angiogenic agent, an anti-mitotic agent, an anthracycline, toxin,and an apoptotic agent.

In another embodiment the binding protein described above is acrystallized binding protein and exists as a crystal. Preferably thecrystal is a carrier-free pharmaceutical controlled release crystal.More preferably the crystallized binding protein has a greater half lifein vivo than the soluble counterpart of said binding protein. Mostpreferably the crystallized binding protein retains biological activity.

In another embodiment the binding protein described above isglycosylated. Preferably the glycosylation is a human glycosylationpattern.

One aspect of the invention pertains to an isolated nucleic acidencoding any one of the binding protein disclosed above. A furtherembodiment provides a vector comprising the isolated nucleic aciddisclosed above wherein said vector is selected from the groupconsisting of pcDNA; pTT (Durocher et al., Nucleic Acids Research 2002,Vol 30, No. 2); pTT3 (pTT with additional multiple cloning site; pEFBOS(Mizushima, S, and Nagata, S., (1990) Nucleic acids Research Vol 18, No.17); pBV; pJV; pcDNA3.1 TOPO®, pEF6 TOPO® and pBJ.

In another aspect a host cell is transformed with the vector disclosedabove. Preferably the host cell is a prokaryotic cell. More preferablythe host cell is E. Coli. In a related embodiment the host cell is aneukaryotic cell. Preferably the eukaryotic cell is selected from thegroup consisting of protist cell, animal cell, plant cell and fungalcell. More preferably the host cell is a mammalian cell including, butnot limited to, CHO and COS; or a fungal cell such as Saccharomycescerevisiae; or an insect cell such as Sf9.

Another aspect of the invention provides a method of producing a bindingprotein disclosed above comprising culturing any one of the host cellsalso disclosed above in a culture medium under conditions sufficient toproduce the binding protein. Preferably 50%-75% of the binding proteinproduced by this method is a dual specific tetravalent binding protein.More preferably 75%-90% of the binding protein produced by this methodis a dual specific tetravalent binding protein. Most preferably 90%-95%of the binding protein produced is a dual specific tetravalent bindingprotein.

Another embodiment provides a binding protein produced according to themethod disclosed above.

One embodiment provides a composition for the release of a bindingprotein wherein the composition comprises a formulation which in turncomprises a crystallized binding protein, as disclosed above and aningredient; and at least one polymeric carrier. Preferably the polymericcarrier is a polymer selected from one or more of the group consistingof: poly (acrylic acid), poly (cyanoacrylates), poly (amino acids), poly(anhydrides), poly (depsipeptide), poly (esters), poly (lactic acid),poly (lactic-co-glycolic acid) or PLGA, poly (b-hydroxybutryate), poly(caprolactone), poly (dioxanone); poly (ethylene glycol), poly((hydroxypropyl)methacrylamide, poly [(organo)phosphazene], poly (orthoesters), poly (vinyl alcohol), poly (vinylpyrrolidone), maleicanhydride-alkyl vinyl ether copolymers, pluronic polyols, albumin,alginate, cellulose and cellulose derivatives, collagen, fibrin,gelatin, hyaluronic acid, oligosaccharides, glycaminoglycans, sulfatedpolyeaccharides, blends and copolymers thereof. Preferably theingredient is selected from the group consisting of albumin, sucrose,trehalose, lactitol, gelatin, hydroxypropyl-β-cyclodextrin,methoxypolyethylene glycol and polyethylene glycol. Another embodimentprovides a method for treating a mammal comprising the step ofadministering to the mammal an effective amount of the compositiondisclosed above.

The invention also provides a pharmaceutical composition comprising abinding protein, as disclosed above and a pharmaceutically acceptablecarrier. In a further embodiment the pharmaceutical compositioncomprises at least one additional therapeutic agent for treating adisorder. Preferably the additional agent is selected from the groupconsisting of: Therapeutic agent, imaging agent, cytotoxic agent,angiogenesis inhibitors (including but not limited to anti-VEGFantibodies or VEGF-trap); kinase inhibitors (including but not limitedto KDR and TIE-2 inhibitors); co-stimulation molecule blockers(including but not limited to anti-B7.1, anti-B7.2, CTLA4-Ig,anti-CD20); adhesion molecule blockers (including but not limited toanti-LFA-1 Abs, anti-E/L selectin Abs, small molecule inhibitors);anti-cytokine antibody or functional fragment thereof (including but notlimited to anti-IL-18, anti-TNF, anti-IL-6/cytokine receptorantibodies); methotrexate; cyclosporin; rapamycin; FK506; detectablelabel or reporter; a TNF antagonist; an antirheumatic; a musclerelaxant, a narcotic, a non-steroid anti-inflammatory drug (NSAID), ananalgesic, an anesthetic, a sedative, a local anesthetic, aneuromuscular blocker, an antimicrobial, an antipsoriatic, acorticosteriod, an anabolic steroid, an erythropoietin, an immunization,an immunoglobulin, an immunosuppressive, a growth hormone, a hormonereplacement drug, a radiopharmaceutical, an antidepressant, anantipsychotic, a stimulant, an asthma medication, a beta agonist, aninhaled steroid, an epinephrine or analog, a cytokine, and a cytokineantagonist.

In another aspect, the invention provides a method for treating a humansubject suffering from a disorder in which the target, or targets,capable of being bound by the binding protein disclosed above isdetrimental, comprising administering to the human subject a bindingprotein disclosed above such that the activity of the target, or targetsin the human subject is inhibited and treatment is achieved. Preferablythe disorder is selected from the group comprising arthritis,osteoarthritis, juvenile chronic arthritis, septic arthritis, Lymearthritis, psoriatic arthritis, reactive arthritis, spondyloarthropathy,systemic lupus erythematosus, Crohn's disease, ulcerative colitis,inflammatory bowel disease, insulin dependent diabetes mellitus,thyroiditis, asthma, allergic diseases, psoriasis, dermatitisscleroderma, graft versus host disease, organ transplant rejection,acute or chronic immune disease associated with organ transplantation,sarcoidosis, atherosclerosis, disseminated intravascular coagulation,Kawasaki's disease, Grave's disease, nephrotic syndrome, chronic fatiguesyndrome, Wegener's granulomatosis, Henoch-Schoenlein purpurea,microscopic vasculitis of the kidneys, chronic active hepatitis,uveitis, septic shock, toxic shock syndrome, sepsis syndrome, cachexia,infectious diseases, parasitic diseases, acquired immunodeficiencysyndrome, acute transverse myelitis, Huntington's chorea, Parkinson'sdisease, Alzheimer's disease, stroke, primary biliary cirrhosis,hemolytic anemia, malignancies, heart failure, myocardial infarction,Addison's disease, sporadic, polyglandular deficiency type I andpolyglandular deficiency type II, Schmidt's syndrome, adult (acute)respiratory distress syndrome, alopecia, alopecia greata, seronegativearthopathy, arthropathy, Reiter's disease, psoriatic arthropathy,ulcerative colitic arthropathy, enteropathic synovitis, chlamydia,yersinia and salmonella associated arthropathy, spondyloarthopathy,atheromatous disease/arteriosclerosis, atopic allergy, autoimmunebullous disease, pemphigus vulgaris, pemphigus foliaceus, pemphigoid,linear IgA disease, autoimmune haemolytic anaemia, Coombs positivehaemolytic anaemia, acquired pernicious anaemia, juvenile perniciousanaemia, myalgic encephalitis/Royal Free Disease, chronic mucocutaneouscandidiasis, giant cell arteritis, primary sclerosing hepatitis,cryptogenic autoimmune hepatitis, Acquired Immunodeficiency DiseaseSyndrome, Acquired Immunodeficiency Related Diseases, Hepatitis B,Hepatitis C, common varied immunodeficiency (common variablehypogammaglobulinaemia), dilated cardiomyopathy, female infertility,ovarian failure, premature ovarian failure, fibrotic lung disease,cryptogenic fibrosing alveolitis, post-inflammatory interstitial lungdisease, interstitial pneumonitis, connective tissue disease associatedinterstitial lung disease, mixed connective tissue disease associatedlung disease, systemic sclerosis associated interstitial lung disease,rheumatoid arthritis associated interstitial lung disease, systemiclupus erythematosus associated lung disease,dermatomyositis/polymyositis associated lung disease, Sjögren's diseaseassociated lung disease, ankylosing spondylitis associated lung disease,vasculitic diffuse lung disease, haemosiderosis associated lung disease,drug-induced interstitial lung disease, fibrosis, radiation fibrosis,bronchiolitis obliterans, chronic eosinophilic pneumonia, lymphocyticinfiltrative lung disease, postinfectious interstitial lung disease,gouty arthritis, autoimmune hepatitis, type-1 autoimmune hepatitis(classical autoimmune or lupoid hepatitis), type-2 autoimmune hepatitis(anti-LKM antibody hepatitis), autoimmune mediated hypoglycaemia, type Binsulin resistance with acanthosis nigricans, hypoparathyroidism, acuteimmune disease associated with organ transplantation, chronic immunedisease associated with organ transplantation, osteoarthrosis, primarysclerosing cholangitis, psoriasis type 1, psoriasis type 2, idiopathicleucopaenia, autoimmune neutropaenia, renal disease NOS,glomerulonephritides, microscopic vasulitis of the kidneys, lymedisease, discoid lupus erythematosus, male infertility idiopathic orNOS, sperm autoimmunity, multiple sclerosis (all subtypes), sympatheticophthalmia, pulmonary hypertension secondary to connective tissuedisease, Goodpasture's syndrome, pulmonary manifestation ofpolyarteritis nodosa, acute rheumatic fever, rheumatoid spondylitis,Still's disease, systemic sclerosis, Sjörgren's syndrome, Takayasu'sdisease/arteritis, autoimmune thrombocytopaenia, idiopathicthrombocytopaenia, autoimmune thyroid disease, hyperthyroidism, goitrousautoimmune hypothyroidism (Hashimoto's disease), atrophic autoimmunehypothyroidism, primary myxoedema, phacogenic uveitis, primaryvasculitis, vitiligo acute liver disease, chronic liver diseases,alcoholic cirrhosis, alcohol-induced liver injury, choleosatatis,idiosyncratic liver disease, Drug-Induced hepatitis, Non-alcoholicSteatohepatitis, allergy and asthma, group B streptococci (GBS)infection, mental disorders (e.g., depression and schizophrenia), Th2Type and Th1 Type mediated diseases, acute and chronic pain (differentforms of pain), and cancers such as lung, breast, stomach, bladder,colon, pancreas, ovarian, prostate and rectal cancer and hematopoieticmalignancies (leukemia and lymphoma), Abetalipoprotemia, Acrocyanosis,acute and chronic parasitic or infectious processes, acute leukemia,acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), acuteor chronic bacterial infection, acute pancreatitis, acute renal failure,adenocarcinomas, aerial ectopic beats, AIDS dementia complex,alcohol-induced hepatitis, allergic conjunctivitis, allergic contactdermatitis, allergic rhinitis, allograft rejection, alpha-1-antitrypsindeficiency, amyotrophic lateral sclerosis, anemia, angina pectoris,anterior horn cell degeneration, anti cd3 therapy, antiphospholipidsyndrome, anti-receptor hypersensitivity reactions, aordic andperipheral aneuryisms, aortic dissection, arterial hypertension,arteriosclerosis, arteriovenous fistula, ataxia, atrial fibrillation(sustained or paroxysmal), atrial flutter, atrioventricular block, Bcell lymphoma, bone graft rejection, bone marrow transplant (BMT)rejection, bundle branch block, Burkitt's lymphoma, Burns, cardiacarrhythmias, cardiac stun syndrome, cardiac tumors, cardiomyopathy,cardiopulmonary bypass inflammation response, cartilage transplantrejection, cerebellar cortical degenerations, cerebellar disorders,chaotic or multifocal atrial tachycardia, chemotherapy associateddisorders, chromic myelocytic leukemia (CML), chronic alcoholism,chronic inflammatory pathologies, chronic lymphocytic leukemia (CLL),chronic obstructive pulmonary disease (COPD), chronic salicylateintoxication, colorectal carcinoma, congestive heart failure,conjunctivitis, contact dermatitis, cor pulmonale, coronary arterydisease, Creutzfeldt-Jakob disease, culture negative sepsis, cysticfibrosis, cytokine therapy associated disorders, Dementia pugilistica,demyelinating diseases, dengue hemorrhagic fever, dermatitis,dermatologic conditions, diabetes, diabetes mellitus, diabeticateriosclerotic disease, Diffuse Lewy body disease, dilated congestivecardiomyopathy, disorders of the basal ganglia, Down's Syndrome inmiddle age, drug-induced movement disorders induced by drugs which blockCNS dopamine receptors, drug sensitivity, eczema, encephalomyelitis,endocarditis, endocrinopathy, epiglottitis, epstein-barr virusinfection, erythromelalgia, extrapyramidal and cerebellar disorders,familial hematophagocytic lymphohistiocytosis, fetal thymus implantrejection, Friedreich's ataxia, functional peripheral arterialdisorders, fungal sepsis, gas gangrene, gastric ulcer, glomerularnephritis, graft rejection of any organ or tissue, gram negative sepsis,gram positive sepsis, granulomas due to intracellular organisms, hairycell leukemia, Hallerrorden-Spatz disease, hashimoto's thyroiditis, hayfever, heart transplant rejection, hemachromatosis, hemodialysis,hemolytic uremic syndrome/thrombolytic thrombocytopenic purpura,hemorrhage, hepatitis (A), His bundle arrythmias, HIV infection/HIVneuropathy, Hodgkin's disease, hyperkinetic movement disorders,hypersensitity reactions, hypersensitivity pneumonitis, hypertension,hypokinetic movement disorders, hypothalamic-pituitary-adrenal axisevaluation, idiopathic Addison's disease, idiopathic pulmonary fibrosis,antibody mediated cytotoxicity, Asthenia, infantile spinal muscularatrophy, inflammation of the aorta, influenza a, ionizing radiationexposure, iridocyclitis/uveitis/optic neuritis, ischemia-reperfusioninjury, ischemic stroke, juvenile rheumatoid arthritis, juvenile spinalmuscular atrophy, Kaposi's sarcoma, kidney transplant rejection,legionella, leishmaniasis, leprosy, lesions of the corticospinal system,lipedema, liver transplant rejection, lymphederma, malaria, malignamtLymphoma, malignant histiocytosis, malignant melanoma, meningitis,meningococcemia, metabolic/idiopathic, migraine headache, mitochondrialmulti.system disorder, mixed connective tissue disease, monoclonalgammopathy, multiple myeloma, multiple systems degenerations (MencelDejerine-Thomas Shi-Drager and Machado-Joseph), myasthenia gravis,mycobacterium avium intracellulare, mycobacterium tuberculosis,myelodyplastic syndrome, myocardial infarction, myocardial ischemicdisorders, nasopharyngeal carcinoma, neonatal chronic lung disease,nephritis, nephrosis, neurodegenerative diseases, neurogenic I muscularatrophies, neutropenic fever, non-hodgkins lymphoma, occlusion of theabdominal aorta and its branches, occulsive arterial disorders, okt3therapy, orchitis/epidydimitis, orchitis/vasectomy reversal procedures,organomegaly, osteoporosis, pancreas transplant rejection, pancreaticcarcinoma, paraneoplastic syndrome/hypercalcemia of malignancy,parathyroid transplant rejection, pelvic inflammatory disease, perennialrhinitis, pericardial disease, peripheral atherlosclerotic disease,peripheral vascular disorders, peritonitis, pernicious anemia,pneumocystis carinii pneumonia, pneumonia, POEMS syndrome(polyneuropathy, organomegaly, endocrinopathy, monoclonal gammopathy,and skin changes syndrome), post perfusion syndrome, post pump syndrome,post-MI cardiotomy syndrome, preeclampsia, Progressive supranucleoPalsy, primary pulmonary hypertension, radiation therapy, Raynaud'sphenomenon and disease, Raynoud's disease, Refsum's disease, regularnarrow QRS tachycardia, renovascular hypertension, reperfusion injury,restrictive cardiomyopathy, sarcomas, scleroderma, senile chorea, SenileDementia of Lewy body type, seronegative arthropathies, shock, sicklecell anemia, skin allograft rejection, skin changes syndrome, smallbowel transplant rejection, solid tumors, specific arrythmias, spinalataxia, spinocerebellar degenerations, streptococcal myositis,structural lesions of the cerebellum, Subacute sclerosingpanencephalitis, Syncope, syphilis of the cardiovascular system,systemic anaphalaxis, systemic inflammatory response syndrome, systemiconset juvenile rheumatoid arthritis, T-cell or FAB ALL, Telangiectasia,thromboangitis obliterans, thrombocytopenia, toxicity, transplants,trauma/hemorrhage, type III hypersensitivity reactions, type IVhypersensitivity, unstable angina, uremia, urosepsis, urticaria,valvular heart diseases, varicose veins, vasculitis, venous diseases,venous thrombosis, ventricular fibrillation, viral and fungalinfections, vital encephalitis/aseptic meningitis, vital-associatedhemaphagocytic syndrome, Wernicke-Korsakoff syndrome, Wilson's disease,xenograft rejection of any organ or tissue.

In another aspect the invention provides a method of treating a patientsuffering from a disorder comprising the step of administering any oneof the binding proteins disclosed above before, concurrent, or after theadministration of a second agent, as discussed above. In a preferredembodiment the second agent is selected from the group consisting ofbudenoside, epidermal growth factor, corticosteroids, cyclosporin,sulfasalazine, aminosalicylates, 6-mercaptopurine, azathioprine,metronidazole, lipoxygenase inhibitors, mesalamine, olsalazine,balsalazide, antioxidants, thromboxane inhibitors, IL-1 receptorantagonists, anti-IL-1β monoclonal antibodies, anti-IL-6 monoclonalantibodies, growth factors, elastase inhibitors, pyridinyl-imidazolecompounds, antibodies or agonists of TNF, LT, IL-1, IL-2, IL-6, IL-7,IL-8, IL-12, IL-13, IL-15, IL-16, IL-18, IL-23, EMAP-II, GM-CSF, FGF,and PDGF, antibodies of CD2, CD3, CD4, CD8, CD-19, CD25, CD28, CD30,CD40, CD45, CD69, CD90 or their ligands, methotrexate, cyclosporin,FK506, rapamycin, mycophenolate mofetil, leflunomide, NSAIDs, ibuprofen,corticosteroids, prednisolone, phosphodiesterase inhibitors, adensosineagonists, antithrombotic agents, complement inhibitors, adrenergicagents, IRAK, NIK, IKK, p38, MAP kinase inhibitors, IL-1β convertingenzyme inhibitors, TNFαconverting enzyme inhibitors, T-cell signallinginhibitors, metalloproteinase inhibitors, sulfasalazine, azathioprine,6-mercaptopurines, angiotensin converting enzyme inhibitors, solublecytokine receptors, soluble p55 TNF receptor, soluble p75 TNF receptor,sIL-1RI, sIL-1RII, sIL-6R, antiinflammatory cytokines, IL-4, IL-10,IL-11, IL-13 and TGFβ.

In a preferred embodiment the pharmaceutical compositions disclosedabove are administered to the subject by at least one mode selected fromparenteral, subcutaneous, intramuscular, intravenous, intrarticular,intrabronchial, intraabdominal, intracapsular, intracartilaginous,intracavitary, intracelial, intracerebellar, intracerebroventricular,intracolic, intracervical, intragastric, intrahepatic, intramyocardial,intraosteal, intrapelvic, intrapericardiac, intraperitoneal,intrapleural, intraprostatic, intrapulmonary, intrarectal, intrarenal,intraretinal, intraspinal, intrasynovial, intrathoracic, intrauterine,intravesical, bolus, vaginal, rectal, buccal, sublingual, intranasal,and transdermal.

One aspect of the invention provides at least one anti-idiotype antibodyto at least one binding protein of the present invention. Theanti-idiotype antibody includes any protein or peptide containingmolecule that comprises at least a portion of an immunoglobulin moleculesuch as, but not limited to, at least one complementarily determiningregion (CDR) of a heavy or light chain or a ligand binding portionthereof, a heavy chain or light chain variable region, a heavy chain orlight chain constant region, a framework region, or; any portionthereof, that can be incorporated into a binding protein of the presentinvention.

In another embodiment the binding proteins of the invention are capableof binding one or more targets selected from the group consisting ofABCF1; ACVR1; ACVR1B; ACVR2; ACVR2B; ACVRL1; ADORA2A; Aggrecan; AGR2;AICDA; AIF1; AIG1; AKAP1; AKAP2; AMH; AMHR2; ANGPT1; ANGPT2; ANGPTL3;ANGPTL4; ANPEP; APC; APOC1; AR; AZGP1 (zinc-a-glycoprotein); B7.1; B7.2;BAD; BAFF; BAG1; BAI1; BCL2; BCL6; BDNF; BLNK; BLR1 (MDR15); BlyS; BMP1;BMP2; BMP3B (GDF10); BMP4; BMP6; BMP8; BMPRIA; BMPR1B; BMPR2; BPAG1(plectin); BRCA1; C19orf10 (IL27w); C3; C4A; C5; C5R1; CANT1; CASP1;CASP4; CAV1; CCBP2 (D6/JAB61); CCL1 (1-309); CCL11 (eotaxin); CCL13(MCP-4); CCL15 (MIP-1d); CCL16 (HCC-4); CCL17 (TARC); CCL18 (PARC);CCL19 (MIP-3b); CCL2 (MCP-1); MCAF; CCL20 (MIP-3a); CCL21 (MIP-2); SLC;exodus-2; CCL22 (MDC/STC-1); CCL23 (MPIF-1); CCL24 (MPIF-2/eotaxin-2);CCL25 (TECK); CCL26 (eotaxin-3); CCL27 (CTACK/ILC); CCL28; CCL3(MIP-1a); CCL4 (MIP-1b); CCL5 (RANTES); CCL7 (MCP-3); CCL8 (mcp-2);CCNA1; CCNA2; CCND1; CCNE1; CCNE2; CCR1 (CKR1/HM 145); CCR2(mcp-1RB/RA); CCR3 (CKR3/CMKBR3); CCR4; CCR5 (CMKBR5/ChemR13); CCR6(CMKBR6/CKR-L3/STRL22/DRY6); CCR7 (CKR7/EB11); CCR8(CMKBR8/TER1/CKR-L1); CCR9 (GPR-9-6); CCRL1 (VSHK1); CCRL2 (L-CCR);CD164; CD19; CD1C; CD20; CD200; CD-22; CD24; CD28; CD3; CD37; CD38;CD3E; CD3G; CD3Z; CD4; CD40; CD40L; CD44; CD45RB; CD52; CD69; CD72;CD74; CD79A; CD79B; CD8; CD80; CD81; CD83; CD86; CDH1 (E-cadherin);CDH10; CDH12; CDH13; CDH18; CDH19; CDH20; CDH5; CDH7; CDH8; CDH9; CDK2;CDK3; CDK4; CDK5; CDK6; CDK7; CDK9; CDKN1A (p21Wap1/Cip1); CDKN1B(p27Kip1); CDKN1C; CDKN2A (p161NK4a); CDKN2B; CDKN2C; CDKN3; CEBPB;CER1; CHGA; CHGB; Chitinase; CHST10; CKLFSF2; CKLFSF3; CKLFSF4; CKLFSF5;CKLFSF6; CKLFSF7; CKLFSF8; CLDN3; CLDN7 (claudin-7); CLN3; CLU(clusterin); CMKLR1; CMKOR1 (RDC1); CNR1; COL18A1; COL1A1; COL4A3;COL6A1; CR2; CRP; CSF1 (M-CSF); CSF2 (GM-CSF); CSF3 (GCSF); CTLA4;CTNNB1 (b-catenin); CTSB (cathepsin B); CX3CL1 (SCYD1); CX3CR1 (V28);CXCL1 (GRO1); CXCL10 (IP-10); CXCL11 (1-TAC/IP-9); CXCL12 (SDF1);CXCL13; CXCL14; CXCL16; CXCL2 (GRO2); CXCL3 (GRO3); CXCL5 (ENA-78/LIX);CXCL6 (GCP-2); CXCL9 (MIG); CXCR3 (GPR9/CKR-L2); CXCR4; CXCR6(TYMSTR/STRL33/Bonzo); CYB5; CYC1; CYSLTR1; DAB2IP; DES; DKFZp451J0118;DNCL1; DPP4; E2F1; ECGF1; EDG1; EFNA1; EFNA3; EFNB2; EGF; EGFR; ELAC2;ENG; ENO1; ENO2; ENO3; EPHB4; EPO; ERBB2 (Her-2); EREG; ERK8; ESR1;ESR2; F3 (TF); FADD; FasL; FASN; FCER1A; FCER2; FCGR3A; FGF; FGF1(aFGF); FGF10; FGF11; FGF12; FGF12B; FGF13; FGF14; FGF16; FGF17; FGF18;FGF19; FGF2 (bFGF); FGF20; FGF21; FGF22; FGF23; FGF3 (int-2); FGF4(HST); FGF5; FGF6 (HST-2); FGF7 (KGF); FGF8; FGF9; FGFR3; FIGF (VEGFD);FIL1 (EPSILON); FIL1 (ZETA); FLJ12584; FLJ25530; FLRT1 (fibronectin);FLT1; FOS; FOSL1 (FRA-1); FY (DARC); GABRP (GABAa); GAGEB1; GAGEC1;GALNAC4S-6ST; GATA3; GDF5; GFI1; GGT1; GM-CSF; GNAS1; GNRH1; GPR2(CCR10); GPR31; GPR44; GPR81 (FKSG80); GRCC10 (C10); GRP; GSN(Gelsolin); GSTP1; HAVCR2; HDAC4; HDAC5; HDAC7A; HDAC9; HGF; HIF1A;HIP1; histamine and histamine receptors; HLA-A; HLA-DRA; HM74; HMOX1;HUMCYT2A; ICEBERG; ICOSL; ID2; IFN-a; IFNA1; IFNA2; IFNA4; IFNA5; IFNA6;IFNA7; IFNB1; IFNgamma; IFNW1; IGBP1; IGF1; IGF1R; IGF2; IGFBP2; IGFBP3;IGFBP6; IL-1; IL10; IL10RA; IL10RB; IL11; IL11RA; IL-12; IL12A; IL12B;IL12RB1; IL12RB2; IL13; IL13RA1; IL13RA2; IL14; IL15; IL15RA; IL16;IL17; IL17B; IL17C; IL17R; IL18; IL18BP; IL18R1; IL18RAP; IL19; IL1A;IL1B; IL1F10; IL1F5; IL1F6; IL1F7; IL1F8; IL1F9; IL1HY1; IL1R1; IL1R2;IL1RAP; IL1RAPL1; IL1RAPL2; IL1RL1; IL1RL2 IL1RN; IL2; IL20; IL20RA;IL21R; IL22; IL22R; IL22RA2; IL23; IL24; IL25; IL26; IL27; IL28A; IL28B;IL29; IL2RA; IL2RB; IL2RG; IL3; IL30; IL3RA; IL4; IL4R; IL5; IL5RA; IL6;IL6R; IL6ST (glycoprotein 130); IL7; IL7R; IL8; IL8RA; IL8RB; IL8RB;IL9; IL9R; ILK; INHA; INHBA; INSL3; INSL4; IRAK1; IRAK2; ITGA1; ITGA2;ITGA3; ITGA6 (a6 integrin); ITGAV; ITGB3; ITGB4 (b 4 integrin); JAG1;JAK1; JAK3; JUN; K6HF; KAI1; KDR; KITLG; KLF5 (GC Box BP); KLF6; KLK10;KLK12; KLK13; KLK14; KLK15; KLK3; KLK4; KLK5; KLK6; KLK9; KRT1; KRT19(Keratin 19); KRT2A; KRTHB6 (hair-specific type II keratin); LAMA5; LEP(leptin); Lingo-p75; Lingo-Troy; LPS; LTA (TNF-b); LTB; LTB4R (GPR16);LTB4R2; LTBR; MACMARCKS; MAG or Omgp; MAP2K7 (c-Jun); MDK; MIB1;midkine; MIF; MIP-2; MKI67 (Ki-67); MMP2; MMP9; MS4A1; MSMB; MT3(metallothionectin-III); MTSS1; MUC1 (mucin); MYC; MYD88; NCK2;neurocan; NFKB1; NFKB2; NGFB (NGF); NGFR; NgR-Lingo; NgR-Nogo66 (Nogo);NgR-p75; NgR-Troy; NME1 (NM23A); NOX5; NPPB; NR0B1; NR0B2; NR1D1; NR1D2;NR1H2; NR1H3; NR1H4; NR1I2; NR1I3; NR2C1; NR2C2; NR2E1; NR2E3; NR2F1;NR2F2; NR2F6; NR3C1; NR3C2; NR4A1; NR4A2; NR4A3; NR5A1; NR5A2; NR6A1;NRP1; NRP2; NT5E; NTN4; ODZ1; OPRD1; P2Rx7; PAP; PART1; PATE; PAWR;PCA3; PCNA; PDGFA; PDGFB; PECAM1; PF4 (CXCL4); PGF; PGR; phosphacan;PIAS2; PIK3CG; PLAU (uPA); PLG; PLXDC1; PPBP (CXCL7); PPID; PR1; PRKCQ;PRKD1; PRL; PROC; PROK2; PSAP; PSCA; PTAFR; PTEN; PTGS2 (COX-2); PTN;RAC2 (p21Rac2); RARB; RGS1; RGS13; RGS3; RNF110 (ZNF144); ROBO2; S100A2;SCGB1D2 (lipophilin B); SCGB2A1 (mammaglobin 2); SCGB2A2 (mammaglobin1); SCYE1 (endothelial Monocyte-activating cytokine); SDF2; SERPINA1;SERPINA3; SERPINB5 (maspin); SERPINE1 (PAI-1); SERPINF1; SHBG; SLA2;SLC2A2; SLC33A1; SLC43A1; SLIT2; SPP1; SPRR1B (Spr1); ST6GAL1; STAB1;STAT6; STEAP; STEAP2; TB4R2; TBX21; TCP10; TDGF1; TEK; TGFA; TGFB1;TGFB11; TGFB2; TGFB3; TGFB1; TGFBR1; TGFBR2; TGFBR3; TH1L; THBS1(thrombospondin-1); THBS2; THBS4; THPO; TIE (Tie-1); TIMP3; tissuefactor; TLR10; TLR2; TLR3; TLR4; TLR5; TLR6; TLR7; TLR8; TLR9; TNF;TNF-a; TNFAIP2 (B94); TNFAIP3; TNFRSF1A; TNFRSF1A; TNFRSF1B; TNFRSF21;TNFRSF5; TNFRSF6 (Fas); TNFRSF7; TNFRSF8; TNFRSF9; TNFSF10 (TRAIL);TNFSF11 (TRANCE); TNFSF12 (APO3L); TNFSF13 (April); TNFSF13B; TNFSF14(HVEM-L); TNFSF15 (VEGI); TNFSF18; TNFSF4 (OX40 ligand); TNFSF5 (CD40ligand); TNFSF6 (FasL); TNFSF7 (CD27 ligand); TNFSF8 (CD30 ligand);TNFSF9 (4-IBB ligand); TOLLIP; Toll-like receptors; TOP2A (topoisomeraseIia); TP53; TPM1; TPM2; TRADD; TRAF1; TRAF2; TRAF3; TRAF4; TRAF5; TRAF6;TREM1; TREM2; TRPC6; TSLP; TWEAK; VEGF; VEGFB; VEGFC; versican; VHL C5;VLA-4; XCL1 (lymphotactin); XCL2 (SCM-1b); XCR1 (GPR5/CCXCR1); YY1; andZFPM2.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A is a schematic representation of Dual Variable Domain (DVD)-Igconstructs and shows the strategy for generation of a DVD-Ig from twoparent antibodies; FIG. 1B, is a schematic representation of constructsDVD1-Ig, DVD2-Ig, and two chimeric mono-specific antibodies fromhybridoma clones 2D13.E3 (anti-IL-1α) and 13F5.G5 (anti-IL-1β).

DETAILED DESCRIPTION OF THE INVENTION

This invention pertains to multivalent and/or multispecific bindingproteins capable of binding two or more antigens. Specifically, theinvention relates to dual variable domain immunoglobulins (DVD-Ig), andpharmaceutical compositions thereof, as well as nucleic acids,recombinant expression vectors and host cells for making such DVD-Igs.Methods of using the DVD-Igs of the invention to detect specificantigens, either in vitro or in vivo are also encompassed by theinvention.

Unless otherwise defined herein, scientific and technical terms used inconnection with the present invention shall have the meanings that arecommonly understood by those of ordinary skill in the art. The meaningand scope of the terms should be clear, however, in the event of anylatent ambiguiy, definitions provided herein take precedent over anydictionary or extrinsic definition. Further, unless otherwise requiredby context, singular terms shall include pluralities and plural termsshall include the singular. In this application, the use of “or” means“and/or” unless stated otherwise. Furthermore, the use of the term“including”, as well as other forms, such as “includes” and “included”,is not limiting. Also, terms such as “element” or “component” encompassboth elements and components comprising one unit and elements andcomponents that comprise more than one subunit unless specificallystated otherwise.

Generally, nomenclatures used in connection with, and techniques of,cell and tissue culture, molecular biology, immunology, microbiology,genetics and protein and nucleic acid chemistry and hybridizationdescribed herein are those well known and commonly used in the art. Themethods and techniques of the present invention are generally performedaccording to conventional methods well known in the art and as describedin various general and more specific references that are cited anddiscussed throughout the present specification unless otherwiseindicated. Enzymatic reactions and purification techniques are performedaccording to manufacturer's specifications, as commonly accomplished inthe art or as described herein. The nomenclatures used in connectionwith, and the laboratory procedures and techniques of, analyticalchemistry, synthetic organic chemistry, and medicinal and pharmaceuticalchemistry described herein are those well known and commonly used in theart. Standard techniques are used for chemical syntheses, chemicalanalyses, pharmaceutical preparation, formulation, and delivery, andtreatment of patients.

That the present invention may be more readily understood, select termsare defined below.

The term “Polypeptide” as used herein, refers to any polymeric chain ofamino acids. The terms “peptide” and “protein” are used interchangeablywith the term polypeptide and also refer to a polymeric chain of aminoacids. The term “polypeptide” encompasses native or artificial proteins,protein fragments and polypeptide analogs of a protein sequence. Apolypeptide may be monomeric or polymeric.

The term “isolated protein” or “isolated polypeptide” is a protein orpolypeptide that by virtue of its origin or source of derivation is notassociated with naturally associated components that accompany it in itsnative state; is substantially free of other proteins from the samespecies; is expressed by a cell from a different species; or does notoccur in nature. Thus, a polypeptide that is chemically synthesized orsynthesized in a cellular system different from the cell from which itnaturally originates will be “isolated” from its naturally associatedcomponents. A protein may also be rendered substantially free ofnaturally associated components by isolation, using protein purificationtechniques well known in the art.

The term “recovering” as used herein, refers to the process of renderinga chemical species such as a polypeptide substantially free of naturallyassociated components by isolation, e.g., using protein purificationtechniques well known in the art.

“Biological activity” as used herein, refers to all inherent biologicalproperties of the antigen. Biological properties include but are notlimited to binding receptor; induction of cell proliferation, inhibitingcell growth, inductions of other cytokines, induction of apoptosis, andenzymatic activity.

The terms “specific binding” or “specifically binding”, as used herein,in reference to the interaction of an antibody, a protein, or a peptidewith a second chemical species, mean that the interaction is dependentupon the presence of a particular structure (e.g., an antigenicdeterminant or epitope) on the chemical species; for example, anantibody recognizes and binds to a specific protein structure ratherthan to proteins generally. If an antibody is specific for epitope “A”,the presence of a molecule containing epitope A (or free, unlabeled A),in a reaction containing labeled “A” and the antibody, will reduce theamount of labeled A bound to the antibody.

The term “antibody”, as used herein, broadly refers to anyimmunoglobulin (Ig) molecule comprised of four polypeptide chains, twoheavy (H) chains and two light (L) chains, or any functional fragment,mutant, variant, or derivation thereof, which retains the essentialepitope binding features of an Ig molecule. Such mutant, variant, orderivative antibody formats are known in the art. Nonlimitingembodiments of which are discussed below.

In a full-length antibody, each heavy chain is comprised of a heavychain variable region (abbreviated herein as HCVR or VH) and a heavychain constant region. The heavy chain constant region is comprised ofthree domains, CH1, CH2 and CH3. Each light chain is comprised of alight chain variable region (abbreviated herein as LCVR or VL) and alight chain constant region. The light chain constant region iscomprised of one domain, CL. The VH and VL regions can be furthersubdivided into regions of hypervariability, termed complementaritydetermining regions (CDR), interspersed with regions that are moreconserved, termed framework regions (FR). Each VH and VL is composed ofthree CDRs and four FRs, arranged from amino-terminus tocarboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3,CDR3, FR4. Immunoglobulin molecules can be of any type (e.g., IgG, IgE,IgM, IgD, IgA and IgY), class (e.g., IgG 1, IgG2, IgG 3, IgG4, IgA1 andIgA2) or subclass.

The term “Fc region” is used to define the C-terminal region of animmunoglobulin heavy chain, which may be generated by papain digestionof an intact antibody. The Fc region may be a native sequence Fc regionor a variant Fc region. The Fc region of an immunoglobulin generallycomprises two constant domains, a CH2 domain and a CH3 domain, andoptionally comprises a CH4 domain. Replacements of amino acid residuesin the Fc portion to alter antibody effector function are known in theart (Winter, et al. U.S. Pat. Nos. 5,648,260; 5,624,821). The Fc portionof an antibody mediates several important effector functions e.g.cytokine induction, ADCC, phagocytosis, complement dependentcytotoxicity (CDC) and half-life/clearance rate of antibody andantigen-antibody complexes. In some cases these effector functions aredesirable for therapeutic antibody but in other cases might beunnecessary or even deleterious, depending on the therapeuticobjectives. Certain human IgG isotypes, particularly IgG1 and IgG3,mediate ADCC and CDC via binding to FcγRs and complement C1q,respectively. Neonatal Fc receptors (FcRn) are the critical componentsdetermining the circulating half-life of antibodies. In still anotherembodiment at least one amino acid residue is replaced in the constantregion of the antibody, for example the Fc region of the antibody, suchthat effector functions of the antibody are altered. The dimerization oftwo identical heavy chains of an immunoglobulin is mediated by thedimerization of CH3 domains and is stabilized by the disulfide bondswithin the hinge region (Huber et al. Nature; 264: 415-20; Thies et al1999 J Mol Biol; 293: 67-79.). Mutation of cysteine residues within thehinge regions to prevent heavy chain-heavy chain disulfide bonds willdestabilize dimeration of CH3 domains. Residues responsible for CH3dimerization have been identified (Dall'Acqua 1998 Biochemistry 37:9266-73.). Therefore, it is possible to generate a monovalent half-Ig.Interestingly, these monovalent half Ig molecules have been found innature for both IgG and IgA subclasses (Seligman 1978 Ann Immunol 129:855-70; Biewenga et al 1983 Clin Exp Immunol 51: 395-400). Thestoichiometry of FcRn: Ig Fc region has been determined to be 2:1 (Westet al 2000 Biochemistry 39: 9698-708), and half Fc is sufficient formediating FcRn binding (Kim et al 1994 Eur J Immunol; 24: 542-548.).Mutations to disrupt the dimerization of CH3 domain may not have greateradverse effect on its FcRn binding as the residues important for CH3dimerization are located on the inner interface of CH3 b sheetstructure, whereas the region responsible for FcRn binding is located onthe outside interface of CH2-CH3 domains. However the half Ig moleculemay have certain advantage in tissue penetration due to its smaller sizethan that of a regular antibody. In one embodiment at least one aminoacid residue is replaced in the constant region of the binding proteinof the invention, for example the Fc region, such that the dimerizationof the heavy chains is disrupted, resulting in half DVD Ig molecules.

The term “antigen-binding portion” of an antibody (or simply “antibodyportion”), as used herein, refers to one or more fragments of anantibody that retain the ability to specifically bind to an antigen. Ithas been shown that the antigen-binding function of an antibody can beperformed by fragments of a full-length antibody. Such antibodyembodiments may also be bispecific, dual specific, or multi-specificformats; specifically binding to two or more different antigens.Examples of binding fragments encompassed within the term“antigen-binding portion” of an antibody include (i) a Fab fragment, amonovalent fragment consisting of the VL, VH, CL and CH1 domains; (ii) aF(ab′)₂ fragment, a bivalent fragment comprising two Fab fragmentslinked by a disulfide bridge at the hinge region; (iii) a Fd fragmentconsisting of the VH and CH1 domains; (iv) a Fv fragment consisting ofthe VL and VH domains of a single arm of an antibody, (v) a dAb fragment(Ward et al., (1989) Nature 341:544-546, Winter et al., PCT publicationWO 90/05144 A1 herein incorporated by reference), which comprises asingle variable domain; and (vi) an isolated complementarity determiningregion (CDR). Furthermore, although the two domains of the Fv fragment,VL and VH, are coded for by separate genes, they can be joined, usingrecombinant methods, by a synthetic linker that enables them to be madeas a single protein chain in which the VL and VH regions pair to formmonovalent molecules (known as single chain Fv (scFv); see e.g., Bird etal. (1988) Science 242:423-426; and Huston et al. (1988) Proc. Natl.Acad. Sci. USA 85:5879-5883). Such single chain antibodies are alsointended to be encompassed within the term “antigen-binding portion” ofan antibody. Other forms of single chain antibodies, such as diabodiesare also encompassed. Diabodies are bivalent, bispecific antibodies inwhich VH and VL domains are expressed on a single polypeptide chain, butusing a linker that is too short to allow for pairing between the twodomains on the same chain, thereby forcing the domains to pair withcomplementary domains of another chain and creating two antigen bindingsites (see e.g., Holliger, P., et al. (1993) Proc. Natl. Acad. Sci. USA90:6444-6448; Poljak, R. J., et al. (1994) Structure 2:1121-1123). Suchantibody binding portions are known in the art (Kontermann and Dubeleds., Antibody Engineering (2001) Springer-Verlag. New York. 790 pp.(ISBN 3-540-41354-5). In addition single chain antibodies also include“linear antibodies” comprising a pair of tandem Fv segments(VH-CH1-VH-CH1) which, together with complementary light chainpolypeptides, form a pair of antigen binding regions (Zapata et al.Protein Eng. 8(10):1057-1062 (1995); and U.S. Pat. No. 5,641,870).

The term “multivalent binding protein” is used throughout thisspecification to denote a binding protein comprising two or more antigenbinding sites. The multivalent binding protein is preferably engineeredto have the three or more antigen binding sites, and is generally not anaturally occurring antibody. The term “multispecific binding protein”refers to a binding protein capable of binding two or more related orunrelated targets. Dual variable domain (DVD) binding proteins of theinvention comprise two or more antigen binding sites and are tetravalentor multivalent binding proteins. DVDs may be monospecific, i.e capableof binding one antigen or multispecific, i.e. capable of binding two ormore antigens. DVD binding proteins comprising two heavy chain DVDpolypeptides and two light chain DVD polypeptides are referred to a DVDIg. Each half of a DVD Ig comprises a heavy chain DVD polypeptide, and alight chain DVD polypeptide, and two antigen binding sites. Each bindingsite comprises a heavy chain variable domain and a light chain variabledomain with a total of 6 CDRs involved in antigen binding per antigenbinding site.

The term “bispecific antibody”, as used herein, refers to full-lengthantibodies that are generated by quadroma technology (see Milstein, C.and A. C. Cuello, Nature, 1983. 305(5934): p. 537-40), by chemicalconjugation of two different mAbs (see Staerz, U. D., et al., Nature,1985. 314(6012): p. 628-31), or by knob-into-hole or similar approacheswhich introduces mutations in the Fc region (see Holliger, P., T.Prospero, and G. Winter, Proc Natl Acad Sci USA, 1993. 90(14): p.6444-8.18), resulting in multiple different immunogloblin species ofwhich only one is the functional bispecific antibody. By molecularfunction, a bispecific antibody binds one antigen (or epitope) on one ofits two binding arms (one pair of HC/LC), and binds a different antigen(or epitope) on its second arm (a different pair of HC/LC). By thisdefinition, a bispecific antibody has two distinct antigen binding arms(in both specificity and CDR sequences), and is mono-valent for eachantigen it binds to.

The term “dual-specific antibody”, as used herein, refers to full-lengthantibodies that can bind two different antigens (or epitopes) in each ofits two binding arms (a pair of HC/LC) (see PCT publication WO02/02773). Accordingly a dual-specific binding protein has two identicalantigen binding arms, with identical specificity and identical CDRsequences, and is bi-valent for each antigen it binds to.

A “functional antigen binding site” of a binding protein is one which iscapable of binding a target antigen. The antigen binding affinity of theantigen binding site is not necessarily as strong as the parent antibodyfrom which the antigen binding site is derived, but the ability to bindantigen must be measurable using any one of a variety of methods knownfor evaluating antibody binding to an antigen. Moreover, the antigenbinding affinity of each of the antigen binding sites of a multivalentantibody herein need not be quantitatively the same.

The term “cytokine” is a generic term for proteins released by one cellpopulation, which act on another cell population as intercellularmediators. Examples of such cytokines are lymphokines, monokines, andtraditional polypeptide hormones. Included among the cytokines aregrowth hormone such as human growth hormone, N-methionyl human growthhormone, and bovine growth hormone; parathyroid hormone; thyroxine;insulin; proinsulin; relaxin; prorelaxin; glycoprotein hormones such asfollicle stimulating hormone (FSH), thyroid stimulating hormone (TSH),and luteinizing hormone (LH); hepatic growth factor; fibroblast growthfactor; prolactin; placental lactogen; tumor necrosis factor-alpha and-beta; mullerian-inhibiting substance; mouse gonadotropin-associatedpeptide; inhibin; activin; vascular endothelial growth factor; integrin;thrombopoietin (TPO); nerve growth factors such as NGF-alpha;platelet-growth factor; transforming growth factors (TGFs) such asTGF-alpha and TGF-beta; insulin-like growth factor-1 and -11;erythropoietin (EPO); osteoinductive factors; interferons such asinterferon-alpha, -beta and -gamma colony stimulating factors (CSFs)such as macrophage-CSF (M-CSF); granulocyte macrophage-CSF (GM-CSF); andgranulocyte-CSF (G-CSF); interleukins (ILs) such as IL-1, IL-2, IL-3,IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-15,IL-18, IL-23; a tumor necrosis factor such as TNF-alpha or TNF-beta; andother polypeptide factors including LIF and kit ligand (KL). As usedherein, the term cytokine includes proteins from natural sources or fromrecombinant cell culture and biologically active equivalents of thenative sequence cytokines.

The term “Linker” is used to denote polypeptides comprising two or moreamino acid residues joined by peptide bonds and are used to link one ormore antigen binding portions. Such linker polypeptides are well knownin the art (see e.g., Holliger, P., et al. (1993) Proc. Natl. Acad. Sci.USA 90:6444-6448; Poljak, R. J., et al. (1994) Structure 2:1121-1123).Preferred linker include, but are not limited to, AKTTPKLEEGEFSEAR (SEQID NO: 118); AKTTPKLEEGEFSEARV (SEQ ID NO:119); AKTTPKLGG (SEQ ID NO:120); SAKTTPKLGG (SEQ ID NO: 121); SAKTTP (SEQ ID NO: 122); RADAAP (SEQID NO: 123); RADAAPTVS (SEQ ID NO: 124); RADAAAAGGPGS (SEQ ID NO: 125);RADAAAA(G₄S)₄ (SEQ ID NO: 126); SAKTTPKLEEGEFSEARV (SEQ ID NO: 127);ADAAP (SEQ ID NO:40); ADAAPTVSIFPP (SEQ ID NO: 103); TVAAP (SEQ IDNO:44); TVAAPSVFIFPP (SEQ ID NO:50); QPKAAP (SEQ ID NO:88);QPKAAPSVTLFPP (SEQ ID NO:92); AKTTPP (SEQ ID NO:38); AKTTPPSVTPLAP (SEQID NO: 128); AKTTAP (SEQ ID NO: 129); AKTTAPSVYPLAP (SEQ ID NO:99);ASTKGP (SEQ ID NO:42); ASTKGPSVFPLAP (SEQ ID NO:48).

An immunoglobulin constant domain refers to a heavy or light chainconstant domain. Human IgG heavy chain and light chain constant domainamino acid sequences are known in the art.

The term “monoclonal antibody” as used herein refers to an antibodyobtained from a population of substantially homogeneous antibodies,i.e., the individual antibodies comprising the population are identicalexcept for possible naturally occurring mutations that may be present inminor amounts. Monoclonal antibodies are highly specific, being directedagainst a single antigen. Furthermore, in contrast to polyclonalantibody preparations that typically include different antibodiesdirected against different determinants (epitopes), each monoclonalantibody is directed against a single determinant on the antigen. Themodifier “monoclonal” is not to be construed as requiring production ofthe antibody by any particular method.

The term “human antibody”, as used herein, is intended to includeantibodies having variable and constant regions derived from humangermline immunoglobulin sequences. The human antibodies of the inventionmay include amino acid residues not encoded by human germlineimmunoglobulin sequences (e.g., mutations introduced by random orsite-specific mutagenesis in vitro or by somatic mutation in vivo), forexample in the CDRs and in particular CDR3. However, the term “humanantibody”, as used herein, is not intended to include antibodies inwhich CDR sequences derived from the germline of another mammalianspecies, such as a mouse, have been grafted onto human frameworksequences.

The term “recombinant human antibody”, as used herein, is intended toinclude all human antibodies that are prepared, expressed, created orisolated by recombinant means, such as antibodies expressed using arecombinant expression vector transfected into a host cell (describedfurther in Section II C, below), antibodies isolated from a recombinant,combinatorial human antibody library (Hoogenboom H. R., (1997) TIB Tech.15:62-70; Azzazy H., and Highsmith W. E., (2002) Clin. Biochem.35:425-445; Gavilondo J. V., and Larrick J. W. (2002) BioTechniques29:128-145; Hoogenboom H., and Chames P. (2000) Immunology Today21:371-378), antibodies isolated from an animal (e.g., a mouse) that istransgenic for human immunoglobulin genes (see e.g., Taylor, L. D., etal. (1992) Nucl. Acids Res. 20:6287-6295; Kellermann S-A., and Green L.L. (2002) Current Opinion in Biotechnology 13:593-597; Little M. et al(2000) Immunology Today 21:364-370) or antibodies prepared, expressed,created or isolated by any other means that involves splicing of humanimmunoglobulin gene sequences to other DNA sequences. Such recombinanthuman antibodies have variable and constant regions derived from humangermline immunoglobulin sequences. In certain embodiments, however, suchrecombinant human antibodies are subjected to in vitro mutagenesis (or,when an animal transgenic for human Ig sequences is used, in vivosomatic mutagenesis) and thus the amino acid sequences of the VH and VLregions of the recombinant antibodies are sequences that, while derivedfrom and related to human germline VH and VL sequences, may notnaturally exist within the human antibody germline repertoire in vivo.

An “affinity matured” antibody is one with one or more alterations inone or more CDRs thereof which result an improvement in the affinity ofthe antibody for antigen, compared to a parent antibody which does notpossess those alteration(s). Preferred affinity matured antibodies willhave nanomolar or even picomolar affinities for the target antigen.Affinity matured antibodies are produced by procedures known in the art.Marks et al. BidlTechnology 10:779-783 (1992) describes affinitymaturation by VH and VL domain shuffling. Random mutagenesis of CDRand/or framework residues is described by: Barbas et al. Proc Nat. Acad.Sci, USA 91:3809-3813 (1994); Schier et al. Gene 169:147-155 (1995);Yelton et al. J. Immunol. 155:1994-2004 (1995); Jackson et al., J.Immunol. 154(7):3310-9 (1995); and Hawkins et al, J. Mol. Biol.226:889-896 (1992).

The term “chimeric antibody” refers to antibodies which comprise heavyand light chain variable region sequences from one species and constantregion sequences from another species, such as antibodies having murineheavy and light chain variable regions linked to human constant regions.

The term “CDR-grafted antibody” refers to antibodies which compriseheavy and light chain variable region sequences from one species but inwhich the sequences of one or more of the CDR regions of VH and/or VLare replaced with CDR sequences of another species, such as antibodieshaving murine heavy and light chain variable regions in which one ormore of the murine CDRs (e.g., CDR3) has been replaced with human CDRsequences.

The term “humanized antibody” refers to antibodies which comprise heavyand light chain variable region sequences from a non-human species(e.g., a mouse) but in which at least a portion of the VH and/or VLsequence has been altered to be more “human-like”, i.e., more similar tohuman germline variable sequences. One type of humanized antibody is aCDR-grafted antibody, in which human CDR sequences are introduced intonon-human VH and VL sequences to replace the corresponding nonhuman CDRsequences.

The terms “Kabat numbering”, “Kabat definitions and “Kabat labeling” areused interchangeably herein. These terms, which are recognized in theart, refer to a system of numbering amino acid residues which are morevariable (i.e. hypervariable) than other amino acid residues in theheavy and light chain variable regions of an antibody, or an antigenbinding portion thereof (Kabat et al. (1971) Ann. NY Acad, Sci.190:382-391 and, Kabat, E. A., et al. (1991) Sequences of Proteins ofImmunological Interest, Fifth Edition, U.S. Department of Health andHuman Services, NIH Publication No. 91-3242). For the heavy chainvariable region, the hypervariable region ranges from amino acidpositions 31 to 35 for CDR1, amino acid positions 50 to 65 for CDR2, andamino acid positions 95 to 102 for CDR3. For the light chain variableregion, the hypervariable region ranges from amino acid positions 24 to34 for CDR1, amino acid positions 50 to 56 for CDR2, and amino acidpositions 89 to 97 for CDR3.

As used herein, the term “CDR” refers to the complementarity determiningregion within antibody variable sequences. There are three CDRs in eachof the variable regions of the heavy chain and the light chain, whichare designated CDR1, CDR2 and CDR3, for each of the variable regions.The term “CDR set” as used herein refers to a group of three CDRs thatoccur in a single variable region capable of binding the antigen. Theexact boundaries of these CDRs have been defined differently accordingto different systems. The system described by Kabat (Kabat et al.,Sequences of Proteins of Immunological Interest (National Institutes ofHealth, Bethesda, Md. (1987) and (1991)) not only provides anunambiguous residue numbering system applicable to any variable regionof an antibody, but also provides precise residue boundaries definingthe three CDRs. These CDRs may be referred to as Kabat CDRs. Chothia andcoworkers (Chothia &Lesk, J. Mol. Biol. 196:901-917 (1987) and Chothiaet al., Nature 342:877-883 (1989)) found that certain sub-portionswithin Kabat CDRs adopt nearly identical peptide backbone conformations,despite having great diversity at the level of amino acid sequence.These sub-portions were designated as L1, L2 and L3 or H1, H2 and H3where the “L” and the “H” designates the light chain and the heavychains regions, respectively. These regions may be referred to asChothia CDRs, which have boundaries that overlap with Kabat CDRs. Otherboundaries defining CDRs overlapping with the Kabat CDRs have beendescribed by Padlan (FASEB J. 9:133-139 (1995)) and MacCallum (J MolBiol 262(5):732-45 (1996)). Still other CDR boundary definitions may notstrictly follow one of the above systems, but will nonetheless overlapwith the Kabat CDRs, although they may be shortened or lengthened inlight of prediction or experimental findings that particular residues orgroups of residues or even entire CDRs do not significantly impactantigen binding. The methods used herein may utilize CDRs definedaccording to any of these systems, although preferred embodiments useKabat or Chothia defined CDRs.

As used herein, the term “framework” or “framework sequence” refers tothe remaining sequences of a variable region minus the CDRs. Because theexact definition of a CDR sequence can be determined by differentsystems, the meaning of a framework sequence is subject tocorrespondingly different interpretations. The six CDRs (CDR-L1, -L2,and -L3 of light chain and CDR-H1, -H2, and -H3 of heavy chain) alsodivide the framework regions on the light chain and the heavy chain intofour sub-regions (FR1, FR2, FR3 and FR4) on each chain, in which CDR1 ispositioned between FR1 and FR2, CDR2 between FR2 and FR3, and CDR3between FR3 and FR4. Without specifying the particular sub-regions asFR1, FR2, FR3 or FR4, a framework region, as referred by others,represents the combined FR's within the variable region of a single,naturally occurring immunoglobulin chain. As used herein, a FRrepresents one of the four sub-regions, and FRs represents two or moreof the four sub-regions constituting a framework region.

As used herein, the term “germline antibody gene” or “gene fragment”refers to an immunoglobulin sequence encoded by non-lymphoid cells thathave not undergone the maturation process that leads to geneticrearrangement and mutation for expression of a particularimmunoglobulin. (See, e.g., Shapiro et al., Crit. Rev. Immunol. 22(3):183-200 (2002); Marchalonis et al., Adv Exp Med Biol. 484:13-30 (2001)).One of the advantages provided by various embodiments of the presentinvention stems from the recognition that germline antibody genes aremore likely than mature antibody genes to conserve essential amino acidsequence structures characteristic of individuals in the species, henceless likely to be recognized as from a foreign source when usedtherapeutically in that species.

As used herein, the term “humanized antibody” is an antibody or avariant, derivative, analog or fragment thereof which immunospecificallybinds to an antigen of interest and which comprises a framework (FR)region having substantially the amino acid sequence of a human antibodyand a complementary determining region (CDR) having substantially theamino acid sequence of a non-human antibody. As used herein, the term“substantially” in the context of a CDR refers to a CDR having an aminoacid sequence at least 80%, preferably at least 85%, at least 90%, atleast 95%, at least 98% or at least 99% identical to the amino acidsequence of a non-human antibody CDR. A humanized antibody comprisessubstantially all of at least one, and typically two, variable domains(Fab, Fab′, F(ab′) 2, FabC, Fv) in which all or substantially all of theCDR regions correspond to those of a non-human immunoglobulin (i.e.,donor antibody) and all or substantially all of the framework regionsare those of a human immunoglobulin consensus sequence. Preferably, ahumanized antibody also comprises at least a portion of animmunoglobulin constant region (Fc), typically that of a humanimmunoglobulin. In some embodiments, a humanized antibody contains boththe light chain as well as at least the variable domain of a heavychain. The antibody also may include the CH1, hinge, CH2, CH3, and CH4regions of the heavy chain. In some embodiments, a humanized antibodyonly contains a humanized light chain. In some embodiments, a humanizedantibody only contains a humanized heavy chain. In specific embodiments,a humanized antibody only contains a humanized variable domain of alight chain and/or humanized heavy chain.

As used herein, the term “neutralizing” refers to neutralization ofbiological activity of a cytokine when a binding protein specificallybinds the cytokine. Preferably the neutralizing binding protein bindsthe cytokine and reduces its biologically activity by at least about20%, 40%, 60%, 80%, 85% or more.

The term “activity” includes activities such as the bindingspecificity/affinity of a DVD-Ig for two or more antigens.

The term “epitope” includes any polypeptide determinant capable ofspecific binding to an immunoglobulin or T-cell receptor. In certainembodiments, epitope determinants include chemically active surfacegroupings of molecules such as amino acids, sugar side chains,phosphoryl, or sulfonyl, and, in certain embodiments, may have specificthree dimensional structural characteristics, and/or specific chargecharacteristics. An epitope is a region of an antigen that is bound byan antibody. In certain embodiments, an antibody is said to specificallybind an antigen when it preferentially recognizes its target antigen ina complex mixture of proteins and/or macromolecules.

The term “surface plasmon resonance”, as used herein, refers to anoptical phenomenon that allows for the analysis of real-time biospecificinteractions by detection of alterations in protein concentrationswithin a biosensor matrix, for example using the BIAcore system(Pharmacia Biosensor AB, Uppsala, Sweden and Piscataway, N.J.). Forfurther descriptions, see Jönsson, U., et al. (1993) Ann. Biol. Clin.51:19-26; Jönsson, U., et al. (1991) Biotechniques 11:620-627; Johnsson,B., et al. (1995) J. Mol. Recognit. 8:125-131; and Johnnson, B., et al.(1991) Anal. Biochem. 198:268-277.

The term “K_(on)”, as used herein, is intended to refer to the on rateconstant for association of an antibody to the antigen to form theantibody/antigen complex as is known in the art.

The term “K_(off)”, as used herein, is intended to refer to the off rateconstant for dissociation of an antibody from the antibody/antigencomplex as is known in the art.

The term “K_(d)”, as used herein, is intended to refer to thedissociation constant of a particular antibody-antigen interaction as isknown in the art.

The term “labeled binding protein” as used herein, refers to a proteinwith a label incorporated that provides for the identification of thebinding protein. Preferably, the label is a detectable marker, e.g.,incorporation of a radiolabeled amino acid or attachment to apolypeptide of biotinyl moieties that can be detected by marked avidin(e.g., streptavidin containing a fluorescent marker or enzymaticactivity that can be detected by optical or colorimetric methods).Examples of labels for polypeptides include, but are not limited to, thefollowing: radioisotopes or radionuclides (e.g., ³H, ¹⁴C, ³⁵S, ⁹⁰Y,⁹⁹Tc, ¹¹¹In, ¹²⁵I, ¹³¹I, ¹⁷⁷Lu, ¹⁶⁶Ho, or ¹⁵³Sm); fluorescent labels(e.g., FITC, rhodamine, lanthanide phosphors), enzymatic labels (e.g.,horseradish peroxidase, luciferase, alkaline phosphatase);chemiluminescent markers; biotinyl groups; predetermined polypeptideepitopes recognized by a secondary reporter (e.g., leucine zipper pairsequences, binding sites for secondary antibodies, metal bindingdomains, epitope tags); and magnetic agents, such as gadoliniumchelates.

The term “conjugate” refers to a binding protein, such as an antibody,chemically linked to a second chemical moiety, such as a therapeutic orcytotoxic agent. The term “agent” is used herein to denote a chemicalcompound, a mixture of chemical compounds, a biological macromolecule,or an extract made from biological materials. Preferably the therapeuticor cytotoxic agents include, but are not limited to, pertussis toxin,taxol, cytochalasin B, gramicidin D, ethidium bromide, emetine,mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicin,doxorubicin, daunorubicin, dihydroxy anthracin dione, mitoxantrone,mithramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids,procaine, tetracaine, lidocaine, propranolol, and puromycin and analogsor homologs thereof.

The terms “crystal”, and “crystallized” as used herein, refer to anantibody, or antigen binding portion thereof, that exists in the form ofa crystal. Crystals are one form of the solid state of matter, which isdistinct from other forms such as the amorphous solid state or theliquid crystalline state. Crystals are composed of regular, repeating,three-dimensional arrays of atoms, ions, molecules (e.g., proteins suchas antibodies), or molecular assemblies (e.g., antigen/antibodycomplexes). These three-dimensional arrays are arranged according tospecific mathematical relationships that are well-understood in thefield. The fundamental unit, or building block, that is repeated in acrystal is called the asymmetric unit. Repetition of the asymmetric unitin an arrangement that conforms to a given, well-definedcrystallographic symmetry provides the “unit cell” of the crystal.Repetition of the unit cell by regular translations in all threedimensions provides the crystal. See Giege, R. and Ducruix, A. Barrett,Crystallization of Nucleic Acids and Proteins, a Practical Approach, 2ndea., pp. 20 1-16, Oxford University Press, New York, N.Y., (1999).”

The term “polynucleotide” as referred to herein means a polymeric formof two or more nucleotides, either ribonucleotides or deoxynucleotidesor a modified form of either type of nucleotide. The term includessingle and double stranded forms of DNA but preferably isdouble-stranded DNA.

The term “isolated polynucleotide” as used herein shall mean apolynucleotide (e.g., of genomic, cDNA, or synthetic origin, or somecombination thereof) that, by virtue of its origin, the “isolatedpolynucleotide”: is not associated with all or a portion of apolynucleotide with which the “isolated polynucleotide” is found innature; is operably linked to a polynucleotide that it is not linked toin nature; or does not occur in nature as part of a larger sequence.

The term “vector”, as used herein, is intended to refer to a nucleicacid molecule capable of transporting another nucleic acid to which ithas been linked. One type of vector is a “plasmid”, which refers to acircular double stranded DNA loop into which additional DNA segments maybe ligated. Another type of vector is a viral vector, wherein additionalDNA segments may be ligated into the viral genome. Certain vectors arecapable of autonomous replication in a host cell into which they areintroduced (e.g., bacterial vectors having a bacterial origin ofreplication and episomal mammalian vectors). Other vectors (e.g.,non-episomal mammalian vectors) can be integrated into the genome of ahost cell upon introduction into the host cell, and thereby arereplicated along with the host genome. Moreover, certain vectors arecapable of directing the expression of genes to which they areoperatively linked. Such vectors are referred to herein as “recombinantexpression vectors” (or simply, “expression vectors”). In general,expression vectors of utility in recombinant DNA techniques are often inthe form of plasmids. In the present specification, “plasmid” and“vector” may be used interchangeably as the plasmid is the most commonlyused form of vector. However, the invention is intended to include suchother forms of expression vectors, such as viral vectors (e.g.,replication defective retroviruses, adenoviruses and adeno-associatedviruses), which serve equivalent functions.

The term “operably linked” refers to a juxtaposition wherein thecomponents described are in a relationship permitting them to functionin their intended manner. A control sequence “operably linked” to acoding sequence is ligated in such a way that expression of the codingsequence is achieved under conditions compatible with the controlsequences. “Operably linked” sequences include both expression controlsequences that are contiguous with the gene of interest and expressioncontrol sequences that act in trans or at a distance to control the geneof interest. The term “expression control sequence” as used hereinrefers to polynucleotide sequences which are necessary to effect theexpression and processing of coding sequences to which they are ligated.Expression control sequences include appropriate transcriptioninitiation, termination, promoter and enhancer sequences; efficient RNAprocessing signals such as splicing and polyadenylation signals;sequences that stabilize cytoplasmic mRNA; sequences that enhancetranslation efficiency (i.e., Kozak consensus sequence); sequences thatenhance protein stability; and when desired, sequences that enhanceprotein secretion. The nature of such control sequences differsdepending upon the host organism; in prokaryotes, such control sequencesgenerally include promoter, ribosomal binding site, and transcriptiontermination sequence; in eukaryotes, generally, such control sequencesinclude promoters and transcription termination sequence. The term“control sequences” is intended to include components whose presence isessential for expression and processing, and can also include additionalcomponents whose presence is advantageous, for example, leader sequencesand fusion partner sequences.

“Transformation”, as defined herein, refers to any process by whichexogenous DNA enters a host cell. Transformation may occur under naturalor artificial conditions using various methods well known in the art.Transformation may rely on any known method for the insertion of foreignnucleic acid sequences into a prokaryotic or eukaryotic host cell. Themethod is selected based on the host cell being transformed and mayinclude, but is not limited to, viral infection, electroporation,lipofection, and particle bombardment. Such “transformed” cells includestably transformed cells in which the inserted DNA is capable ofreplication either as an autonomously replicating plasmid or as part ofthe host chromosome. They also include cells which transiently expressthe inserted DNA or RNA for limited periods of time.

The term “recombinant host cell” (or simply “host cell”), as usedherein, is intended to refer to a cell into which exogenous DNA has beenintroduced. It should be understood that such terms are intended torefer not only to the particular subject cell, but, to the progeny ofsuch a cell. Because certain modifications may occur in succeedinggenerations due to either mutation or environmental influences, suchprogeny may not, in fact, be identical to the parent cell, but are stillincluded within the scope of the term “host cell” as used herein.Preferably host cells include prokaryotic and eukaryotic cells selectedfrom any of the Kingdoms of life. Preferred eukaryotic cells includeprotist, fungal, plant and animal cells. Most preferably host cellsinclude but are not limited to the prokaryotic cell line E. Coli;mammalian cell lines CHO, HEK 293 and COS; the insect cell line Sf9; andthe fungal cell Saccharomyces cerevisiae.

Standard techniques may be used for recombinant DNA, oligonucleotidesynthesis, and tissue culture and transformation (e.g., electroporation,lipofection). Enzymatic reactions and purification techniques may beperformed according to manufacturer's specifications or as commonlyaccomplished in the art or as described herein. The foregoing techniquesand procedures may be generally performed according to conventionalmethods well known in the art and as described in various general andmore specific references that are cited and discussed throughout thepresent specification. See e.g., Sambrook et al. Molecular Cloning: ALaboratory Manual (2d ed., Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y. (1989)), which is incorporated herein by referencefor any purpose.

“Transgenic organism”, as known in the art and as used herein, refers toan organism having cells that contain a transgene, wherein the transgeneintroduced into the organism (or an ancestor of the organism) expressesa polypeptide not naturally expressed in the organism. A “transgene” isa DNA construct, which is stably and operably integrated into the genomeof a cell from which a transgenic organism develops, directing theexpression of an encoded gene product in one or more cell types ortissues of the transgenic organism.

The term “regulate” and “modulate” are used interchangeably, and, asused herein, refers to a change or an alteration in the activity of amolecule of interest (e.g., the biological activity of a cytokine).Modulation may be an increase or a decrease in the magnitude of acertain activity or function of the molecule of interest. Exemplaryactivities and functions of a molecule include, but are not limited to,binding characteristics, enzymatic activity, cell receptor activation,and signal transduction.

Correspondingly, the term “modulator,” as used herein, is a compoundcapable of changing or altering an activity or function of a molecule ofinterest (e.g., the biological activity of a cytokine). For example, amodulator may cause an increase or decrease in the magnitude of acertain activity or function of a molecule compared to the magnitude ofthe activity or function observed in the absence of the modulator. Incertain embodiments, a modulator is an inhibitor, which decreases themagnitude of at least one activity or function of a molecule. Exemplaryinhibitors include, but are not limited to, proteins, peptides,antibodies, peptibodies, carbohydrates or small organic molecules.Peptibodies are described, e.g., in WO01/83525.

The term “agonist”, as used herein, refers to a modulator that, whencontacted with a molecule of interest, causes an increase in themagnitude of a certain activity or function of the molecule compared tothe magnitude of the activity or function observed in the absence of theagonist. Particular agonists of interest may include, but are notlimited to, polypeptides, nucleic acids, carbohydrates, or any othermolecules that bind to the antigen.

The term “antagonist” or “inhibitor”, as used herein, refer to amodulator that, when contacted with a molecule of interest causes adecrease in the magnitude of a certain activity or function of themolecule compared to the magnitude of the activity or function observedin the absence of the antagonist. Particular antagonists of interestinclude those that block or modulate the biological or immunologicalactivity of the antigen. Antagonists and inhibitors of antigens mayinclude, but are not limited to, proteins, nucleic acids, carbohydrates,or any other molecules, which bind to the antigen.

As used herein, the term “effective amount” refers to the amount of atherapy which is sufficient to reduce or ameliorate the severity and/orduration of a disorder or one or more symptoms thereof, prevent theadvancement of a disorder, cause regression of a disorder, prevent therecurrence, development, onset or progression of one or more symptomsassociated with a disorder, detect a disorder, or enhance or improve theprophylactic or therapeutic effect(s) of another therapy (e.g.,prophylactic or therapeutic agent).

The term “sample”, as used herein, is used in its broadest sense. A“biological sample”, as used herein, includes, but is not limited to,any quantity of a substance from a living thing or formerly livingthing. Such living things include, but are not limited to, humans, mice,rats, monkeys, dogs, rabbits and other animals. Such substances include,but are not limited to, blood, serum, urine, synovial fluid, cells,organs, tissues, bone marrow, lymph nodes and spleen.

I. Generation of DVD Binding Protein

The invention pertains to Dual Variable Domain binding proteins capableof binding one or more targets and methods of making the same.Preferably the binding protein comprises a polypeptide chain, whereinsaid polypeptide chain comprises VD1-(X1)n-VD2-C—(X2)n, wherein VD1 is afirst variable domain, VD2 is a second variable domain, C is a constantdomain, X1 represents an amino acid or polypeptide, X2 represents an Fcregion and n is 0 or 1. The binding protein of the invention can begenerated using various techniques. The invention provides expressionvectors, host cell and methods of generating the binding protein.

A. Generation of Parent Monoclonal Antibodies

The variable domains of the DVD binding protein can be obtained fromparent antibodies, including polyclonal and monoclonal antibodiescapable of binding antigens of interest. These antibodies may benaturally occurring or may be generated by recombinant technology.

Monoclonal antibodies can be prepared using a wide variety of techniquesknown in the art including the use of hybridoma, recombinant, and phagedisplay technologies, or a combination thereof. For example, monoclonalantibodies can be produced using hybridoma techniques including thoseknown in the art and taught, for example, in Harlow et al., Antibodies:A Laboratory Manual, (Cold Spring Harbor Laboratory Press, 2nd ed.1988); Hammerling, et al., in: Monoclonal Antibodies and T-CellHybridomas 563-681 (Elsevier, N.Y., 1981) (said references incorporatedby reference in their entireties). The term “monoclonal antibody” asused herein is not limited to antibodies produced through hybridomatechnology. The term “monoclonal antibody” refers to an antibody that isderived from a single clone, including any eukaryotic, prokaryotic, orphage clone, and not the method by which it is produced. Hybridomas areselected, cloned and further screened for desirable characteristics,including robust hybridoma growth, high antibody production anddesirable antibody characteristics, as discussed in Example 1 below.Hybridomas may be cultured and expanded in vivo in syngeneic animals, inanimals that lack an immune system, e.g., nude mice, or in cell culturein vitro. Methods of selecting, cloning and expanding hybridomas arewell known to those of ordinary skill in the art. In a preferredembodiment, the hybridomas are mouse hybridomas. In another preferredembodiment, the hybridomas are produced in a non-human, non-mousespecies such as rats, sheep, pigs, goats, cattle or horses. In anotherembodiment, the hybridomas are human hybridomas, in which a humannon-secretory myeloma is fused with a human cell expressing an antibodycapable of binding a specific antigen.

Recombinant monoclonal antibodies are also generated from single,isolated lymphocytes using a procedure referred to in the art as theselected lymphocyte antibody method (SLAM), as described in U.S. Pat.No. 5,627,052, PCT Publication WO 92/02551 and Babcock, J. S. et al.(1996) Proc. Natl. Acad. Sci. USA 93:7843-7848. In this method, singlecells secreting antibodies of interest, e.g., lymphocytes derived froman immunized animal, are identified, and, heavy- and light-chainvariable region cDNAs are rescued from the cells by reversetranscriptase-PCR and these variable regions can then be expressed, inthe context of appropriate immunoglobulin constant regions (e.g., humanconstant regions), in mammalian host cells, such as COS or CHO cells.The host cells transfected with the amplified immunoglobulin sequences,derived from in vivo selected lymphocytes, can then undergo furtheranalysis and selection in vitro, for example by panning the transfectedcells to isolate cells expressing antibodies to the antigen of interest.The amplified immunoglobulin sequences further can be manipulated invitro, such as by in vitro affinity maturation methods such as thosedescribed in PCT Publication WO 97/29131 and PCT Publication WO00/56772.

Monoclonal antibodies are also produced by immunizing a non-human animalcomprising some, or all, of the human immunoglobulin locus with anantigen of interest. In a preferred embodiment, the non-human animal isa XENOMOUSE® transgenic mouse, an engineered mouse strain that compriseslarge fragments of the human immunoglobulin loci and is deficient inmouse antibody production. See, e.g., Green et al. Nature Genetics7:13-21 (1994) and U.S. Pat. Nos. 5,916,771, 5,939,598, 5,985,615,5,998,209, 6,075,181, 6,091,001, 6,114,598 and 6,130,364. See also WO91/10741, published Jul. 25, 1991, WO 94/02602, published Feb. 3, 1994,WO 96/34096 and WO 96/33735, both published Oct. 31, 1996, WO 98/16654,published Apr. 23, 1998, WO 98/24893, published Jun. 11, 1998, WO98/50433, published Nov. 12, 1998, WO 99/45031, published Sep. 10, 1999,WO 99/53049, published Oct. 21, 1999, WO 00 09560, published Feb. 24,2000 and WO 00/037504, published Jun. 29, 2000. The XENOMOUSE®transgenic mouse produces an adult-like human repertoire of fully humanantibodies, and generates antigen-specific human Mabs. The XENOMOUSE®transgenic mouse contains approximately 80% of the human antibodyrepertoire through introduction of megabase sized, germlineconfiguration YAC fragments of the human heavy chain loci and x lightchain loci. See Mendez et al., Nature Genetics 15:146-156 (1997), Greenand Jakobovits J. Exp. Med. 188:483-495 (1998), the disclosures of whichare hereby incorporated by reference.

In vitro methods also can be used to make the parent antibodies, whereinan antibody library is screened to identify an antibody having thedesired binding specificity. Methods for such screening of recombinantantibody libraries are well known in the art and include methodsdescribed in, for example, Ladner et al. U.S. Pat. No. 5,223,409; Kanget al. PCT Publication No. WO 92/18619; Dower et al. PCT Publication No.WO 91/17271; Winter et al. PCT Publication No. WO 92/20791; Markland etal. PCT Publication No. WO 92/15679; Breitling et al. PCT PublicationNo. WO 93/01288; McCafferty et al. PCT Publication No. WO 92/01047;Garrard et al. PCT Publication No. WO 92/09690; Fuchs et al. (1991)Bio/Technology 9:1370-1372; Hay et al. (1992) Hum Antibod Hybridomas3:81-85; Huse et al. (1989) Science 246:1275-1281; McCafferty et al.,Nature (1990) 348:552-554; Griffiths et al. (1993) EMBO J 12:725-734;Hawkins et al. (1992) J Mol Biol 226:889-896; Clackson et al. (1991)Nature 352:624-628; Gram et al. (1992) PNAS 89:3576-3580; Garrad et al.(1991) Bio/Technology 9:1373-1377; Hoogenboom et al. (1991) Nuc Acid Res19:4133-4137; and Barbas et al. (1991) PNAS 88:7978-7982, US patentapplication publication 20030186374, and PCT Publication No. WO97/29131, the contents of each of which are incorporated herein byreference.

Parent antibodies of the present invention can also be generated usingvarious phage display methods known in the art. In phage displaymethods, functional antibody domains are displayed on the surface ofphage particles which carry the polynucleotide sequences encoding them.In a particular, such phage can be utilized to display antigen-bindingdomains expressed from a repertoire or combinatorial antibody library(e.g., human or murine). Phage expressing an antigen binding domain thatbinds the antigen of interest can be selected or identified withantigen, e.g., using labeled antigen or antigen bound or captured to asolid surface or bead. Phage used in these methods are typicallyfilamentous phage including fd and M13 binding domains expressed fromphage with Fab, Fv or disulfide stabilized Fv antibody domainsrecombinantly fused to either the phage gene III or gene VIII protein.Examples of phage display methods that can be used to make theantibodies of the present invention include those disclosed in Brinkmanet al., J. Immunol. Methods 182:41-50 (1995); Ames et al., J. Immunol.Methods 184:177-186 (1995); Kettleborough et al., Eur. J. Immunol.24:952-958 (1994); Persic et al., Gene 187 9-18 (1997); Burton et al.,Advances in Immunology 57:191-280 (1994); PCT application No.PCT/GB91/01134; PCT publications WO 90/02809; WO 91/10737; WO 92/01047;WO 92/18619; WO 93/11236; WO 95/15982; WO 95/20401; and U.S. Pat. Nos.5,698,426; 5,223,409; 5,403,484; 5,580,717; 5,427,908; 5,750,753;5,821,047; 5,571,698; 5,427,908; 5,516,637; 5,780,225; 5,658,727;5,733,743 and 5,969,108; each of which is incorporated herein byreference in its entirety.

As described in the above references, after phage selection, theantibody coding regions from the phage can be isolated and used togenerate whole antibodies including human antibodies or any otherdesired antigen binding fragment, and expressed in any desired host,including mammalian cells, insect cells, plant cells, yeast, andbacteria, e.g., as described in detail below. For example, techniques torecombinantly produce Fab, Fab′ and F(ab′)2 fragments can also beemployed using methods known in the art such as those disclosed in PCTpublication WO 92/22324; Mullinax et al., BioTechniques 12(6):864-869(1992); and Sawai et al., AJRI 34:26-34 (1995); and Better et al.,Science 240:1041-1043 (1988) (said references incorporated by referencein their entireties). Examples of techniques which can be used toproduce single-chain Fvs and antibodies include those described in U.S.Pat. Nos. 4,946,778 and 5,258,498; Huston et al., Methods in Enzymology203:46-88 (1991); Shu et al., PNAS 90:7995-7999 (1993); and Skerra etal., Science 240:1038-1040 (1988).

Alternative to screening of recombinant antibody libraries by phagedisplay, other methodologies known in the art for screening largecombinatorial libraries can be applied to the identification of parentantibodies. One type of alternative expression system is one in whichthe recombinant antibody library is expressed as RNA-protein fusions, asdescribed in PCT Publication No. WO 98/31700 by Szostak and Roberts, andin Roberts, R. W. and Szostak, J. W. (1997) Proc. Natl. Acad. Sci. USA94:12297-12302. In this system, a covalent fusion is created between anmRNA and the peptide or protein that it encodes by in vitro translationof synthetic mRNAs that carry puromycin, a peptidyl acceptor antibiotic,at their 3′ end. Thus, a specific mRNA can be enriched from a complexmixture of mRNAs (e.g., a combinatorial library) based on the propertiesof the encoded peptide or protein, e.g., antibody, or portion thereof,such as binding of the antibody, or portion thereof, to the dualspecificity antigen. Nucleic acid sequences encoding antibodies, orportions thereof, recovered from screening of such libraries can beexpressed by recombinant means as described above (e.g., in mammalianhost cells) and, moreover, can be subjected to further affinitymaturation by either additional rounds of screening of mRNA-peptidefusions in which mutations have been introduced into the originallyselected sequence(s), or by other methods for affinity maturation invitro of recombinant antibodies, as described above.

In another approach the parent antibodies can also be generated usingyeast display methods known in the art. In yeast display methods,genetic methods are used to tether antibody domains to the yeast cellwall and display them on the surface of yeast. In particular, such yeastcan be utilized to display antigen-binding domains expressed from arepertoire or combinatorial antibody library (e.g., human or murine).Examples of yeast display methods that can be used to make the parentantibodies include those disclosed in Wittrup, et al. U.S. Pat. No.6,699,658 incorporated herein by reference.

The antibodies described above can be further modified to generate CDRgrafted and Humanized parent antibodies. CDR-grafted parent antibodiescomprise heavy and light chain variable region sequences from a humanantibody wherein one or more of the CDR regions of V_(H) and/or V_(L)are replaced with CDR sequences of murine antibodies capable of bindingantigen of interest. A framework sequence from any human antibody mayserve as the template for CDR grafting. However, straight chainreplacement onto such a framework often leads to some loss of bindingaffinity to the antigen. The more homologous a human antibody is to theoriginal murine antibody, the less likely the possibility that combiningthe murine CDRs with the human framework will introduce distortions inthe CDRs that could reduce affinity. Therefore, it is preferable thatthe human variable framework that is chosen to replace the murinevariable framework apart from the CDRs have at least a 65% sequenceidentity with the murine antibody variable region framework. It is morepreferable that the human and murine variable regions apart from theCDRs have at least 70% sequence identify. It is even more preferablethat the human and murine variable regions apart from the CDRs have atleast 75% sequence identity. It is most preferable that the human andmurine variable regions apart from the CDRs have at least 80% sequenceidentity. Methods for producing such antibodies are known in the art(see EP 239,400; PCT publication WO 91/09967; U.S. Pat. Nos. 5,225,539;5,530,101; and 5,585,089), veneering or resurfacing (EP 592,106; EP519,596; Padlan, Molecular Immunology 28(4/5):489-498 (1991); Studnickaet al., Protein Engineering 7(6):805-814 (1994); Roguska et al., PNAS91:969-973 (1994)), and chain shuffling (U.S. Pat. No. 5,565,352).

Humanized antibodies are antibody molecules from non-human speciesantibody that binds the desired antigen having one or morecomplementarity determining regions (CDRs) from the non-human speciesand framework regions from a human immunoglobulin molecule. Known humanIg sequences are disclosed, e.g., world wideweb.ncbi.nlm.nih.gov/entrez/query.fcgi; world wideweb.atcc.org/phage/hdb.html; world wide web.sciquest.com/; world wideweb.abcam.com/; world wide web.antibodyresource.com/onlinecomp.html;world wide web.public.iastate.edu/.about.pedro/research_tools.html;world wide web.mgen.uniheidelberg.de/SD/IT/IT.html; world wideweb.whfreeman.com/immunology/CH-05/kuby05.htm; world wideweb.library.thinkquestorg/12429/Immune/Antibody.html; world wideweb.hhmi.org/grants/lectures/1996/vlab/; world wideweb.path.cam.ac.uk/.about.mrc7/mikeimages.html; world wideweb.antibodyresource.com/; mcb.harvard.edu/BioLinks/Immunology.html.world wide web.immunologylink com/;pathbox.wustl.edu/.about.hcenter/index.html; world wideweb.biotech.ufl.eduLabout.hc1/; world wideweb.pebio.com/pa/340913/340913.html-; world wideweb.nal.usda.gov/awic/pubs/antibody/; world wideweb.m.ehimeu.acjp/.about.yasuhito /Elisa.html; world wideweb.biodesign.com/table.asp; world wideweb.icnet.uk/axp/facs/davies/links.html; world wideweb.biotech.ufl.edu/.about.fccl/protocol.html; world wide web.isacnet.org/sites_geo.html;aximtl.imt.unimarburg.de/.about.rek/AEPStart.html;baserv.uci.kun.nl/.about.jraats/linksl.html; world wide web.recab.unihd.de/immuno.bme.nwu.edu/; world wideweb.mrccpe.cam.ac.uk/imtdoc/public/INTRO.html; world wide web.ibt.unam.mx/vir/V_mice.html; imgt.cnusc.fr:8104/; world wideweb.biochem.ucl.ac.uk/.about.martin/abs/index.html;antibody.bath.ac.uk/; abgen.cvm.tamu.edu/lab/world wide web abgen.html;world wide web.unizh.ch/.about.honegger/AHOseminar/Slide01.html; worldwide web.cryst.bbk.ac.uk/.about.ubcg07s/; world wideweb.nimr.mrc.ac.uk/CC/ccaewg/ccaewg.htm; world wideweb.path.cam.ac.uk/.about.mrc7humanisation/TAHHP.html; world wideweb.ibt.unam.mx/vir/structure/stat_aim.html; world wideweb.biosci.missouri.edu/smithgp/index.html; world wideweb.cryst.bioc.cam.ac.uk/.about.fmolina/Webpages/Pept/spottech.html;world wide web.jerini.de/fr roducts.htm; world wideweb.patents.ibm.com/ibm.html.Kabat et al., Sequences of Proteins ofImmunological Interest, U.S. Dept. Health (1983), each entirelyincorporated herein by reference. Such imported sequences can be used toreduce immunogenicity or reduce, enhance or modify binding, affinity,on-rate, off-rate, avidity, specificity, half-life, or any othersuitable characteristic, as known in the art.

Framework residues in the human framework regions may be substitutedwith the corresponding residue from the CDR donor antibody to alter,preferably improve, antigen binding. These framework substitutions areidentified by methods well known in the art, e.g., by modeling of theinteractions of the CDR and framework residues to identify frameworkresidues important for antigen binding and sequence comparison toidentify unusual framework residues at particular positions. (See, e.g.,Queen et al., U.S. Pat. No. 5,585,089; Riechmann et al., Nature 332:323(1988), which are incorporated herein by reference in their entireties.)Three-dimensional immunoglobulin models are commonly available and arefamiliar to those skilled in the art. Computer programs are availablewhich illustrate and display probable three-dimensional conformationalstructures of selected candidate immunoglobulin sequences. Inspection ofthese displays permits analysis of the likely role of the residues inthe functioning of the candidate immunoglobulin sequence, i.e., theanalysis of residues that influence the ability of the candidateimmunoglobulin to bind its antigen. In this way, FR residues can beselected and combined from the consensus and import sequences so thatthe desired antibody characteristic, such as increased affinity for thetarget antigen(s), is achieved. In general, the CDR residues aredirectly and most substantially involved in influencing antigen binding.Antibodies can be humanized using a variety of techniques known in theart, such as but not limited to those described in Jones et al., Nature321:522 (1986); Verhoeyen et al., Science 239:1534 (1988)), Sims et al.,J. Immunol. 151: 2296 (1993); Chothia and Lesk, J. Mol. Biol. 196:901(1987), Carter et al., Proc. Natl. Acad. Sci. U.S.A. 89:4285 (1992);Presta et al., J. Immunol. 151:2623 (1993), Padlan, Molecular Immunology28(4/5):489-498 (1991); Studnicka et al., Protein Engineering7(6):805-814 (1994); Roguska. et al., PNAS 91:969-973 (1994); PCTpublication WO 91/09967, PCT/: US98/16280, US96/18978, US91/09630,US91/05939, US94/01234, GB89/01334, GB91/01134, GB92/01755; WO90/14443,WO90/14424, WO90/14430, EP 229246, EP 592,106; EP 519,596, EP 239,400,U.S. Pat. Nos. 5,565,332, 5,723,323, 5,976,862, 5,824,514, 5,817,483,5,814,476, 5,763,192, 5,723,323, 5,766,886, 5,714,352, 6,204,023,6,180,370, 5,693,762, 5,530,101, 5,585,089, 5,225,539; 4,816,567, eachentirely incorporated herein by reference, included references citedtherein.

Parent monoclonal antibodies may be selected from various monoclonalantibodies capable of binding specific targets and well known in theart. These include, but are not limited to anti-TNF antibody (U.S. Pat.No. 6,258,562), anti-IL-12 and or anti-IL-12p40 antibody (U.S. Pat. No.6,914,128); anti-IL-18 antibody (US 2005/0147610 A1), anti-C5, anti-CBL,anti-CD147, anti-gp120, anti-VLA-4, anti-CD11a, anti-CD18, anti-VEGF,anti-CD40L, anti-Id, anti-ICAM-1, anti-CXCL13, anti-CD2, anti-EGFR,anti-TGF-beta 2, anti-E-selectin, anti-Fact VII, anti-Her2/neu, anti-Fgp, anti-CD11/18, anti-CD14, anti-ICAM-3, anti-CD80, anti-CD4, anti-CD3,anti-CD23, anti-beta2-integrin, anti-alpha4beta7, anti-CD52, anti-HLADR, anti-CD22, anti-CD20, anti-MIF, anti-CD64 (FcR), anti-TCR alphabeta, anti-CD2, anti-Hep B, anti-CA 125, anti-EpCAM, anti-gp120,anti-CMV, anti-gpIIbIIIa, anti-IgE, anti-CD25, anti-CD33, anti-HLA,anti-VNRintegrin, anti-IL-1alpha, anti-IL-1beta, anti-IL-1 receptor,anti-IL-2 receptor, anti-IL-4, anti-IL-4 receptor, anti-IL5, anti-IL-5receptor, anti-IL-6, anti-IL-8, anti-IL-9, anti-IL-13, anti-IL-13receptor, anti-IL-17, and anti-IL-23 (see Presta L G. 2005 Selection,design, and engineering of therapeutic antibodies J Allergy ClinImmunol. 116:731-6 and Clark, M., “Antibodies for TherapeuticApplications,” Department of Pathology, Cambridge University, UK, 15Oct. 2000, published online at M. Clark's home page at the website forthe Department of Pathology, Cambridge University.)

Parent monoclonal antibodies may also be selected from varioustherapeutic antibodies approved for use, in clinical trials, or indevelopment for clinical use. Such therapeutic antibodies include, butare not limited to, rituximab (RITUXAN®, IDEC/Genentech/Roche) (see forexample U.S. Pat. No. 5,736,137), a chimeric anti-CD20 antibody approvedto treat Non-Hodgkin's lymphoma; HUMAX-CD20®, an anti-CD20 currentlybeing developed by Genmab, an anti-CD20 antibody described in U.S. Pat.No. 5,500,362, AME-133 (Applied Molecular Evolution), hA20(Immunomedics, Inc.), HumaLYM (Intracel), and PRO70769(PCT/US2003/040426, entitled “Immunoglobulin Variants and UsesThereof”), trastuzumab (HERCEPTIN®, Genentech) (see for example U.S.Pat. No. 5,677,171), a humanized anti-Her2/neu antibody approved totreat breast cancer; pertuzumab (rhuMab-2C4, OMNITARG®), currently beingdeveloped by Genentech; an anti-Her2 antibody described in U.S. Pat. No.4,753,894; cetuximab (ERB1TUX®, Imclone) (U.S. Pat. No. 4,943,533; PCTWO 96/40210), a chimeric anti-EGFR antibody in clinical trials for avariety of cancers; ABX-EGF (U.S. Pat. No. 6,235,883), currently beingdeveloped by Abgenix-Immunex-Amgen; HUMAX-EGFR™ (U.S. Ser. No.10/172,317), currently being developed by Genmab; 425, EMD55900,EMD62000, and EMD72000 (Merck KGaA) (U.S. Pat. No. 5,558,864; Murthy etal. 1987, Arch Biochem Biophys. 252(2):549-60; Rodeck et al., 1987, JCell Biochem. 35(4):315-20; Kettleborough et al., 1991, Protein Eng.4(7):773-83); ICR62 (Institute of Cancer Research) (PCT WO 95/20045;Modjtahedi et al., 1993, J. Cell Biophys. 1993, 22(1-3):129-46;Modjtahedi et al., 1993, Br J Cancer. 1993, 67(2):247-53; Modjtahedi etal, 1996, Br J Cancer, 73(2):228-35; Modjtahedi et al, 2003, Int JCancer, 105(2):273-80); TheraCIM hR3 (YM Biosciences, Canada and Centrode Immunologia Molecular, Cuba (U.S. Pat. No. 5,891,996; U.S. Pat. No.6,506,883; Mateo et al, 1997, Immunotechnology, 3(1):71-81); mAb-806(Ludwig Institute for Cancer Research, Memorial Sloan-Kettering)(Jungbluth et al. 2003, Proc Natl Acad Sci USA. 100(2):639-44); KSB-102(KS Biomedix); MR1-1 (IVAX, National Cancer Institute) (PCT WO 0162931A2); and SC 100 (Scancell) (PCT WO 01/88138); alemtuzumab (CAMPATH®,Millenium), a humanized monoclonal antibody currently approved fortreatment of B-cell chronic lymphocytic leukemia; muromonab-CD3(Orthoclone OKT3®), an anti-CD3 antibody developed by OrthoBiotech/Johnson & Johnson, ibritumomab tiuxetan (ZEVALIN®), an anti-CD20antibody developed by IDEC/Schering AG, gemtuzumab ozogamicin(MYLOTARG®), an anti-CD33 (p67 protein) antibody developed byCelltech/Wyeth, alefacept (AMEVIVE®), an anti-LFA-3 Fc fusion developedby Biogen), abciximab (REOPRO®), developed by Centocor/Lilly,basiliximab (SIMULECT®), developed by Novartis, palivizumab (SYNAGIS®),developed by Medimmune, infliximab (REMICADE®), an anti-TNFalphaantibody developed by Centocor, adalimumab (HUMIRA®), an anti-TNFalphaantibody developed by Abbott, HUMICADE®, an anti-TNFalpha antibodydeveloped by Celltech, etanercept (ENBREL®), an anti-TNFalpha Fc fusiondeveloped by Immunex/Amgen, ABX-CBL, an anti-CD147 antibody beingdeveloped by Abgenix, ABX-IL8, an anti-IL8 antibody being developed byAbgenix, ABX-MA 1, an anti-MUC18 antibody being developed by Abgenix,Pemtumomab (R1549, 90Y-muHMFG1), an anti-MUC1 in development byAntisoma, Therex (R1550), an anti-MUC1 antibody being developed byAntisoma, AngioMab (AS1405), being developed by Antisoma, HuBC-1, beingdeveloped by Antisoma, Thioplatin (AS1407) being developed by Antisoma,ANTEGREN® (natalizumab), an anti-alpha-4-beta-1 (VLA-4) andalpha-4-beta-7 antibody being developed by Biogen, VLA-1 mAb, ananti-VLA-1 integrin antibody being developed by Biogen, LTBR mAb, ananti-lymphotoxin beta receptor (LTBR) antibody being developed byBiogen, CAT-152, an anti-TGF-β2 antibody being developed by CambridgeAntibody Technology, J695, an anti-IL-12 antibody being developed byCambridge Antibody Technology and Abbott, CAT-192, an anti-TGFβ1antibody being developed by Cambridge Antibody Technology and Genzyme,CAT-213, an anti-Eotaxin1 antibody being developed by Cambridge AntibodyTechnology, LYMPHOSTAT-B® an anti-Blys antibody being developed byCambridge Antibody Technology and Human Genome Sciences Inc.,TRAIL-R1mAb, an anti-TRAIL-R1 antibody being developed by CambridgeAntibody Technology and Human Genome Sciences, Inc., AVASTIN®bevacizumab, rhuMAb-VEGF), an anti-VEGF antibody being developed byGenentech, an anti-HER receptor family antibody being developed byGenentech, Anti-Tissue Factor (ATF), an anti-Tissue Factor antibodybeing developed by Genentech, XOLAIR® (Omalizumab), an anti-IgE antibodybeing developed by Genentech, RAPTIVA® (Efalizumab), an anti-CD11aantibody being developed by Genentech and Xoma, MLN-02 Antibody(formerly LDP-02), being developed by Genentech and MilleniumPharmaceuticals, HUMAX-CD4®, an anti-CD4 antibody being developed byGenmab, HUMAX™-IL15, an anti-IL15 antibody being developed by Genmab andAmgen, HUMAX™-Inflam, being developed by Genmab and Medarex,HUMAX™-Cancer, an anti-Heparanase I antibody being developed by Genmaband Medarex and Oxford GlycoSciences, HUMAX™-Lymphoma, being developedby Genmab and Amgen, HUMAX™-TAC, being developed by Genmab, IDEC-131,and anti-CD40L antibody being developed by IDEC Pharmaceuticals,IDEC-151 (Clenoliximab), an anti-CD4 antibody being developed by IDECPharmaceuticals, IDEC-114, an anti-CD80 antibody being developed by IDECPharmaceuticals, IDEC-152, an anti-CD23 being developed by IDECPharmaceuticals, anti-macrophage migration factor (MIF) antibodies beingdeveloped by IDEC Pharmaceuticals, BEC2, an anti-idiotypic antibodybeing developed by Imclone, IMC-IC11, an anti-KDR antibody beingdeveloped by Imclone, DC101, an anti-flk-1 antibody being developed byImclone, anti-VE cadherin antibodies being developed by Imclone,CEA-CIDE® (labetuzumab), an anti-carcinoembryonic antigen (CEA) antibodybeing developed by Immunomedics, LYMPHOCIDE® (Epratuzumab), an anti-CD22antibody being developed by Immunomedics, AFP-Cide, being developed byImmunomedics, MyelomaCide, being developed by Immunomedics, LkoCide,being developed by Immunomedics, ProstaCide, being developed byImmunomedics, MDX-010, an anti-CTLA4 antibody being developed byMedarex, MDX-060, an anti-CD30 antibody being developed by Medarex,MDX-070 being developed by Medarex, MDX-018 being developed by Medarex,OSIDEM® (IDM-1), and anti-Her2 antibody being developed by Medarex andImmuno-Designed Molecules, HUMAX-CD4®, an anti-CD4 antibody beingdeveloped by Medarex and Genmab, HuMax-IL15, an anti-IL15 antibody beingdeveloped by Medarex and Genmab, CNTO 148, an anti-TNFα antibody beingdeveloped by Medarex and Centocor/J&J, CNTO 1275, an anti-cytokineantibody being developed by Centocor/J&J, MOR101 and MOR102,anti-intercellular adhesion molecule-1 (ICAM-1) (CD54) antibodies beingdeveloped by MorphoSys, MOR201, an anti-fibroblast growth factorreceptor 3 (FGFR-3) antibody being developed by MorphoSys, NUVION®(visilizumab), an anti-CD3 antibody being developed by Protein DesignLabs, HUZAF®, an anti-gamma interferon antibody being developed byProtein Design Labs, Anti-α 5β1 Integrin, being developed by ProteinDesign Labs, anti-IL-12, being developed by Protein Design Labs, ING-1,an anti-Ep-CAM antibody being developed by Xoma, XOLAIR® (Omalizumab) ahumanized anti-IgE antibody developed by Genentech and Novartis, andMLN01, an anti-Beta2 integrin antibody being developed by Xoma, all ofthe above-cited references in this paragraph are expressly incorporatedherein by reference.

B. Construction of DVD Molecules:

The dual variable domain immunoglobulin (DVD-Ig) molecule is designedsuch that two different light chain variable domains (VL) from the twodifferent parent mAbs are linked in tandem directly or via a shortlinker by recombinant DNA techniques, followed by the light chainconstant domain. Similarly, the heavy chain comprises two differentheavy chain variable domains (VH) linked in tandem, followed by theconstant domain CH1 and Fc region (FIG. 1A).

The variable domains can be obtained using recombinant DNA techniquesfrom a parent antibody generated by any one of the methods describedabove. In a preferred embodiment the variable domain is a murine heavyor light chain variable domain. More preferably the variable domain is aCDR grafted or a humanized variable heavy or light chain domain. Mostpreferably the variable domain is a human heavy or light chain variabledomain.

In one embodiment the first and second variable domains are linkeddirectly to each other using recombinant DNA techniques. In anotherembodiment the variable domains are linked via a linker sequence.Preferably two variable domains are linked. Three or more variabledomains may also be linked directly or via a linker sequence. Thevariable domains may bind the same antigen or may bind differentantigens. DVD molecules of the invention may include one immunoglobulinvariable domain and one non-immunoglobulin variable domain such asligand binding domain of a receptor, active domain of an enzyme. DVDmolecules may also comprise 2 or more non-Ig domains.

The linker sequence may be a single amino acid or a polypeptidesequence. Preferably the linker sequences are selected from the groupconsisting of AKTTPKLEEGEFSEAR (SEQ ID NO: 118); AKTTPKLEEGEFSEARV (SEQID NO: 119); AKTTPKLGG (SEQ ID NO: 120); SAKTTPKLGG (SEQ ID NO: 121);SAKTTP (SEQ ID NO: 122); RADAAP (SEQ ID NO: 123); RADAAPTVS (SEQ ID NO:124); RADAAAAGGPGS (SEQ ID NO: 125); RADAAAA(G₄S)₄ (SEQ ID NO: 126);SAKTTPKLEEGEFSEARV (SEQ ID NO: 127); ADAAP (SEQ ID NO:40); ADAAPTVSIFPP(SEQ ID NO: 103); TVAAP (SEQ ID NO:44); TVAAPSVFIFPP (SEQ ID NO:50);QPKAAP (SEQ ID NO:88); QPKAAPSVTLFPP (SEQ ID NO:92); AKTTPP (SEQ IDNO:38); AKTTPPSVTPLAP (SEQ ID NO: 128); AKTTAP (SEQ ID NO: 129);AKTTAPSVYPLAP (SEQ ID NO:99); ASTKGP (SEQ ID NO:42); ASTKGPSVFPLAP (SEQID NO:48); GGGGSGGGGSGGGGS (SEQ ID NO: 130); GENKVEYAPALMALS (SEQ ID NO:131); GPAKELTPLKEAKVS (SEQ ID NO: 132); and GHEAAAVMQVQYPAS (SEQ ID NO:133). The choice of linker sequences is based on crystal structureanalysis of several Fab molecules. There is a natural flexible linkagebetween the variable domain and the CH1/CL constant domain in Fab orantibody molecular structure. This natural linkage comprisesapproximately 10-12 amino acid residues, contributed by 4-6 residuesfrom C-terminus of V domain and 4-6 residues from the N-terminus ofCL/CH1 domain. DVD Igs of the invention were generated using N-terminal5-6 amino acid residues, or 11-12 amino acid residues, of CL or CH1 aslinker in light chain and heavy chain of DVD-Ig, respectively. TheN-terminal residues of CL or CH1 domains, particularly the first 5-6amino acid residues, adopt a loop conformation without strong secondarystructures, therefore can act as flexible linkers between the twovariable domains. The N-terminal residues of CL or CH1 domains arenatural extension of the variable domains, as they are part of the Igsequences, therefore minimize to a large extent any immunogenicitypotentially arising from the linkers and junctions.

Other linker sequences may include any sequence of any length of CL/CH1domain but not all residues of CL/CH1 domain; for example the first 5-12amino acid residues of the CL/CH1 domains; the light chain linkers canbe from Cκ or Cλ; and the heavy chain linkers can be derived from CH1 ofany isotypes, including Cγ1, Cγ2, Cγ3, Cγ4, Cα1, Cα2, Cδ, Cε, and Cμ.Linker sequences may also be derived from other proteins such as Ig-likeproteins, (e.g. TCR, FcR, KIR); G/S based sequences (e.g G4S repeats);hinge region-derived sequences; and other natural sequences from otherproteins.

In a preferred embodiment a constant domain is linked to the two linkedvariable domains using recombinant DNA techniques. Preferably sequencecomprising linked heavy chain variable domains is linked to a heavychain constant domain and sequence comprising linked light chainvariable domains is linked to a light chain constant domain. Preferablythe constant domains are human heavy chain constant domain and humanlight chain constant domain respectively. Most preferably the DVD heavychain is further linked to an Fc region. The Fc region may be a nativesequence Fc region, or a variant Fc region. Most preferably the Fcregion is a human Fc region. In a preferred embodiment the Fc regionincludes Fc region from IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE, or IgD.

In a most preferred embodiment two heavy chain DVD polypeptides and twolight chain DVD polypeptides are combined to form a DVD-Ig molecule.Detailed description of specific DVD-Ig molecules capable of bindingspecific targets, and methods of making the same, is provided in theExamples section below.

C. Production of DVD Proteins

Binding proteins of the present invention may be produced by any of anumber of techniques known in the art. For example, expression from hostcells, wherein expression vector(s) encoding the DVD heavy and DVD lightchains is (are) transfected into a host cell by standard techniques. Thevarious forms of the term “transfection” are intended to encompass awide variety of techniques commonly used for the introduction ofexogenous DNA into a prokaryotic or eukaryotic host cell, e.g.,electroporation, calcium-phosphate precipitation, DEAE-dextrantransfection and the like. Although it is possible to express the DVDproteins of the invention in either prokaryotic or eukaryotic hostcells, expression of DVD proteins in eukaryotic cells is preferable, andmost preferable in mammalian host cells, because such eukaryotic cells(and in particular mammalian cells) are more likely than prokaryoticcells to assemble and secrete a properly folded and immunologicallyactive DVD protein.

Preferred mammalian host cells for expressing the recombinant antibodiesof the invention include Chinese Hamster Ovary (CHO cells) (includingdhfr-CHO cells, described in Urlaub and Chasin, (1980) Proc. Natl. Acad.Sci. USA 77:4216-4220, used with a DHFR selectable marker, e.g., asdescribed in R. J. Kaufman and P. A. Sharp (1982) Mol. Biol.159:601-621), NSO myeloma cells, COS cells and SP2 cells. Whenrecombinant expression vectors encoding DVD proteins are introduced intomammalian host cells, the DVD proteins are produced by culturing thehost cells for a period of time sufficient to allow for expression ofthe DVD proteins in the host cells or, more preferably, secretion of theDVD proteins into the culture medium in which the host cells are grown.DVD proteins can be recovered from the culture medium using standardprotein purification methods.

In a preferred system for recombinant expression of DVD proteins of theinvention, a recombinant expression vector encoding both the DVD heavychain and the DVD light chain is introduced into dhfr-CHO cells bycalcium phosphate-mediated transfection. Within the recombinantexpression vector, the DVD heavy and light chain genes are eachoperatively linked to CMV enhancer/AdMLP promoter regulatory elements todrive high levels of transcription of the genes. The recombinantexpression vector also carries a DHFR gene, which allows for selectionof CHO cells that have been transfected with the vector usingmethotrexate selection/amplification. The selected transformant hostcells are cultured to allow for expression of the DVD heavy and lightchains and intact DVD protein is recovered from the culture medium.Standard molecular biology techniques are used to prepare therecombinant expression vector, transfect the host cells, select fortransformants, culture the host cells and recover the DVD protein fromthe culture medium. Still further the invention provides a method ofsynthesizing a DVD protein of the invention by culturing a host cell ofthe invention in a suitable culture medium until a DVD protein of theinvention is synthesized. The method can further comprise isolating theDVD protein from the culture medium.

An important feature of DVD-Ig is that it can be produced and purifiedin a similar way as a conventional antibody. The production of DVD-Igresults in a homogeneous, single major product with desireddual-specific activity, without any sequence modification of theconstant region or chemical modifications of any kind. Other previouslydescribed methods to generate “bi-specific”, “multi-specific”, and“multi-specific multivalent” full length binding proteins do not lead toa single primary product but instead lead to the intracellular orsecreted production of a mixture of assembled inactive, mono-specific,multi-specific, multivalent, full length binding proteins, andmultivalent full length binding proteins with combination of differentbinding sites. As an example, based on the design described by Millerand Presta (PCT publication WO2001/077342(A1), there are 16 possiblecombinations of heavy and light chains. Consequently only 6.25% ofprotein is likely to be in the desired active form. Separation of fullyactive forms of the protein from inactive and partially active forms ofthe protein using standard chromatography techniques, typically used inlarge scale manufacturing, is yet to be demonstrated.

Surprisingly the design of the “dual-specific multivalent full lengthbinding proteins” of the present invention leads to a dual variabledomain light chain and a dual variable domain heavy chain which assembleprimarily to the desired “dual-specific multivalent full length bindingproteins”.

At least 50%, preferably 75% and more preferably 90% of the assembled,and expressed dual variable domain immunoglobulin molecules are thedesired dual-specific tetravalent protein. This aspect of the inventionparticularly enhances the commercial utility of the invention.Therefore, the present invention includes a method to express a dualvariable domain light chain and a dual variable domain heavy chain in asingle cell leading to a single primary product of a “dual-specifictetravalent full length binding protein”.

The present invention provides a preferred method to express a dualvariable domain light chain and a dual variable domain heavy chain in asingle cell leading to a “primary product” of a “dual-specifictetravalent full length binding protein”, where the “primary product” ismore than 50% of all assembled protein, comprising a dual variabledomain light chain and a dual variable domain heavy chain.

The present invention provides a more preferred method to express a dualvariable domain light chain and a dual variable domain heavy chain in asingle cell leading to a single “primary product” of a “dual-specifictetravalent full length binding protein”, where the “primary product” ismore than 75% of all assembled protein, comprising a dual variabledomain light chain and a dual variable domain heavy chain.

The present invention provides a most preferred method to express a dualvariable domain light chain and a dual variable domain heavy chain in asingle cell leading to a single “primary product” of a “dual-specifictetravalent full length binding protein”, where the “primary product” ismore than 90% of all assembled protein, comprising a dual variabledomain light chain and a dual variable domain heavy chain.

II. Derivatized DVD Binding Proteins:

One embodiment provides a labeled binding protein wherein the bindingprotein of the invention is derivatized or linked to another functionalmolecule (e.g., another peptide or protein). For example, a labeledbinding protein of the invention can be derived by functionally linkingan binding protein of the invention (by chemical coupling, geneticfusion, noncovalent association or otherwise) to one or more othermolecular entities, such as another antibody (e.g., a bispecificantibody or a diabody), a detectable agent, a cytotoxic agent, apharmaceutical agent, and/or a protein or peptide that can mediateassociation of the binding protein with another molecule (such as astreptavidin core region or a polyhistidine tag).

Useful detectable agents with which a binding protein of the inventionmay be derivatized include fluorescent compounds. Exemplary fluorescentdetectable agents include fluorescein, fluorescein isothiocyanate,rhodamine, 5-dimethylamine-1-napthalenesulfonyl chloride, phycoerythrinand the like. A binding protein may also be derivatized with detectableenzymes, such as alkaline phosphatase, horseradish peroxidase, glucoseoxidase and the like. When a binding protein is derivatized with adetectable enzyme, it is detected by adding additional reagents that theenzyme uses to produce a detectable reaction product. For example, whenthe detectable agent horseradish peroxidase is present, the addition ofhydrogen peroxide and diaminobenzidine leads to a colored reactionproduct, which is detectable. a binding protein may also be derivatizedwith biotin, and detected through indirect measurement of avidin orstreptavidin binding.

Another embodiment of the invention provides a crystallized bindingprotein and formulations and compositions comprising such crystals. Inone embodiment the crystallized binding protein has a greater half-lifein vivo than the soluble counterpart of the binding protein. In anotherembodiment the binding protein retains biological activity aftercrystallization.

Crystallized binding protein of the invention may be produced accordingmethods known in the art and as disclosed in WO 02072636, incorporatedherein by reference.

Another embodiment of the invention provides a glycosylated bindingprotein wherein the antibody or antigen-binding portion thereofcomprises one or more carbohydrate residues. Nascent in vivo proteinproduction may undergo further processing, known as post-translationalmodification. In particular, sugar (glycosyl) residues may be addedenzymatically, a process known as glycosylation. The resulting proteinsbearing covalently linked oligosaccharide side chains are known asglycosylated proteins or glycoproteins. Antibodies are glycoproteinswith one or more carbohydrate residues in the Fc domain, as well as thevariable domain. Carbohydrate residues in the Fc domain have importanteffect on the effector function of the Fc domain, with minimal effect onantigen binding or half-life of the antibody (R. Jefferis, Biotechnol.Prog. 21 (2005), pp. 11-16). In contrast, glycosylation of the variabledomain may have an effect on the antigen binding activity of theantibody. Glycosylation in the variable domain may have a negativeeffect on antibody binding affinity, likely due to steric hindrance (Co,M. S., et al., Mol. Immunol. (1993) 30:1361-1367), or result inincreased affinity for the antigen (Wallick, S. C., et al., Exp. Med.(1988) 168:1099-1109; Wright, A., et al., EMBO J. (1991) 10:2717 2723).

One aspect of the present invention is directed to generatingglycosylation site mutants in which the O- or N-linked glycosylationsite of the binding protein has been mutated. One skilled in the art cangenerate such mutants using standard well-known technologies.Glycosylation site mutants that retain the biological activity but haveincreased or decreased binding activity are another object of thepresent invention.

In still another embodiment, the glycosylation of the antibody orantigen-binding portion of the invention is modified. For example, anaglycoslated antibody can be made (i.e., the antibody lacksglycosylation). Glycosylation can be altered to, for example, increasethe affinity of the antibody for antigen. Such carbohydratemodifications can be accomplished by, for example, altering one or moresites of glycosylation within the antibody sequence. For example, one ormore amino acid substitutions can be made that result in elimination ofone or more variable region glycosylation sites to thereby eliminateglycosylation at that site. Such aglycosylation may increase theaffinity of the antibody for antigen. Such an approach is described infurther detail in PCT Publication WO2003016466A2, and U.S. Pat. Nos.5,714,350 and 6,350,861, each of which is incorporated herein byreference in its entirety.

Additionally or alternatively, a modified binding protein of theinvention can be made that has an altered type of glycosylation, such asa hypofucosylated antibody having reduced amounts of fucosyl residues oran antibody having increased bisecting GlcNAc structures. Such alteredglycosylation patterns have been demonstrated to increase the ADCCability of antibodies. Such carbohydrate modifications can beaccomplished by, for example, expressing the antibody in a host cellwith altered glycosylation machinery. Cells with altered glycosylationmachinery have been described in the art and can be used as host cellsin which to express recombinant antibodies of the invention to therebyproduce an antibody with altered glycosylation. See, for example,Shields, R. L. et al. (2002) J. Biol. Chem. 277:26733-26740; Umana etal. (1999) Nat. Biotech. 17:176-1, as well as, European Patent No: EP1,176,195; PCT Publications WO 03/035835; WO 99/54342 80, each of whichis incorporated herein by reference in its entirety.

Protein glycosylation depends on the amino acid sequence of the proteinof interest, as well as the host cell in which the protein is expressed.Different organisms may produce different glycosylation enzymes (eg.,glycosyltransferases and glycosidases), and have different substrates(nucleotide sugars) available. Due to such factors, proteinglycosylation pattern, and composition of glycosyl residues, may differdepending on the host system in which the particular protein isexpressed. Glycosyl residues useful in the invention may include, butare not limited to, glucose, galactose, mannose, fucose,n-acetylglucosamine and sialic acid. Preferably the glycosylated bindingprotein comprises glycosyl residues such that the glycosylation patternis human.

It is known to those skilled in the art that differing proteinglycosylation may result in differing protein characteristics. Forinstance, the efficacy of a therapeutic protein produced in amicroorganism host, such as yeast, and glycosylated utilizing the yeastendogenous pathway may be reduced compared to that of the same proteinexpressed in a mammalian cell, such as a CHO cell line. Suchglycoproteins may also be immunogenic in humans and show reducedhalf-life in vivo after administration. Specific receptors in humans andother animals may recognize specific glycosyl residues and promote therapid clearance of the protein from the bloodstream. Other adverseeffects may include changes in protein folding, solubility,susceptibility to proteases, trafficking, transport,compartmentalization, secretion, recognition by other proteins orfactors, antigenicity, or allergenicity. Accordingly, a practitioner mayprefer a therapeutic protein with a specific composition and pattern ofglycosylation, for example glycosylation composition and patternidentical, or at least similar, to that produced in human cells or inthe species-specific cells of the intended subject animal.

Expressing glycosylated proteins different from that of a host cell maybe achieved by genetically modifying the host cell to expressheterologous glycosylation enzymes. Using techniques known in the art apractitioner may generate antibodies or antigen-binding portions thereofexhibiting human protein glycosylation. For example, yeast strains havebeen genetically modified to express non-naturally occurringglycosylation enzymes such that glycosylated proteins (glycoproteins)produced in these yeast strains exhibit protein glycosylation identicalto that of animal cells, especially human cells (U.S patent applications20040018590 and 20020137134 and PCT publication WO2005100584 A2).

In addition to the binding proteins, the present invention is alsodirected to an anti-idiotypic (anti-Id) antibody specific for suchbinding proteins of the invention. An anti-Id antibody is an antibody,which recognizes unique determinants generally associated with theantigen-binding region of another antibody. The anti-Id can be preparedby immunizing an animal with the binding protein or a CDR containingregion thereof. The immunized animal will recognize, and respond to theidiotypic determinants of the immunizing antibody and produce an anti-Idantibody. The anti-Id antibody may also be used as an “immunogen” toinduce an immune response in yet another animal, producing a so-calledanti-anti-Id antibody.

Further, it will be appreciated by one skilled in the art that a proteinof interest may be expressed using a library of host cells geneticallyengineered to express various glycosylation enzymes, such that memberhost cells of the library produce the protein of interest with variantglycosylation patterns. A practitioner may then select and isolate theprotein of interest with particular novel glycosylation patterns.Preferably, the protein having a particularly selected novelglycosylation pattern exhibits improved or altered biologicalproperties.

III. Uses of DVD-Ig

Given their ability to bind to two or more antigens the binding proteinsof the invention can be used to detect the antigens (e.g., in abiological sample, such as serum or plasma), using a conventionalimmunoassay, such as an enzyme linked immunosorbent assays (ELISA), anradioimmunoassay (RIA) or tissue immunohistochemistry. The DVD-Ig isdirectly or indirectly labeled with a detectable substance to facilitatedetection of the bound or unbound antibody. Suitable detectablesubstances include various enzymes, prosthetic groups, fluorescentmaterials, luminescent materials and radioactive materials. Examples ofsuitable enzymes include horseradish peroxidase, alkaline phosphatase,β-galactosidase, or acetylcholinesterase; examples of suitableprosthetic group complexes include streptavidin/biotin andavidin/biotin; examples of suitable fluorescent materials includeumbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine,dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin; anexample of a luminescent material includes luminol; and examples ofsuitable radioactive material include ³H ¹⁴C, ³⁵S, ⁹⁰Y, ⁹⁹Tc, ¹¹¹In,¹²⁵I, ¹³¹I, ¹⁷⁷Lu, ⁶⁶Ho, or ¹⁵³Sm.

The binding proteins of the invention preferably are capable ofneutralizing the activity of the antigens both in vitro and in vivo.Accordingly, such DVD-Igs can be used to inhibit antigen activity, e.g.,in a cell culture containing the antigens, in human subjects or in othermammalian subjects having the antigens with which a binding protein ofthe invention cross-reacts. In another embodiment, the inventionprovides a method for reducing antigen activity in a subject sufferingfrom a disease or disorder in which the antigen activity is detrimental.A binding protein of the invention can be administered to a humansubject for therapeutic purposes.

As used herein, the term “a disorder in which antigen activity isdetrimental” is intended to include diseases and other disorders inwhich the presence of the antigen in a subject suffering from thedisorder has been shown to be or is suspected of being eitherresponsible for the pathophysiology of the disorder or a factor thatcontributes to a worsening of the disorder. Accordingly, a disorder inwhich antigen activity is detrimental is a disorder in which reductionof antigen activity is expected to alleviate the symptoms and/orprogression of the disorder. Such disorders may be evidenced, forexample, by an increase in the concentration of the antigen in abiological fluid of a subject suffering from the disorder (e.g., anincrease in the concentration of antigen in serum, plasma, synovialfluid, etc. of the subject). Non-limiting examples of disorders that canbe treated with the binding proteins of the invention include thosedisorders discussed below and in the section pertaining topharmaceutical compositions of the antibodies of the invention.

The DVD-Igs of the invention may bind one antigen or multiple antigens.Such antigens include, but are not limited to, the targets listed in thefollowing databases, which databases are incorporated herein byreference. These target databases include those listings:

-   Therapeutic targets (hypertext transfer    protocol://xin.cz3.nus.edu.sg/group/cjttd/ttd.asp);-   Cytokines and cytokine receptors(hypertext transfer    protocol://www.cytokinewebfacts.com/,-   hypertext transfer protocol://www.copewithcytokines.de/cope.cgi, and-   hypertext transfer    protocol://cmbi.bjmu.edu.cn/cmbidata/cgf/CGF_Database/cytokine.medic.-   kumamotou.ac.jp/CFC/indexR.html);-   Chemokines (hypertext transfer    protocol://cytokine.medic.kumamotou.ac.jp/CFC/CK/Chemokine.html);-   Chemokine receptors and GPCRs (hypertext transfer    protocol://csp.medic.kumamotou.ac.jp/CSP/Receptor.html,    http://www.gper.org/7tm/);-   Olfactory Receptors (hypertext transfer    protocol://senselab.med.yale.edu/senselab/ORDB/default. asp);-   Receptors (hypertext transfer    protocol://www.iuphardb.org/iupharrd/list/index.htm);-   Cancer targets (hypertext transfer    protocol://cged.hgc.jp/cgibin/input.cgi);-   Secreted proteins as potential antibody targets (hypertext transfer    protocol://spd.cbi.pku.edu.cn/);-   Protein kinases (hypertext transfer protocol://spd.cbi.pku.edu.cn/),    and-   Human CD markers (hypertext transfer    protocol://contentlabvelocity.com/tools/6/1226/CD_table_final_locked.pdf)    and (Zola H, 2005 CD molecules 2005: human cell differentiation    molecules Blood, 106:3123-6).

DVD-Igs are useful as therapeutic agents to simultaneously block twodifferent targets to enhance efficacy/safety and/or increase patientcoverage. Such targets may include soluble targets (IL-13 and TNF) andcell surface receptor targets (VEGFR and EGFR). It can also be used toinduce redirected cytotoxicity between tumor cells and T cells (Her2 andCD3) for cancer therapy, or between autoreactive cell and effectoe cellsfor autoimmune/transplantation, or between any target cell and effectorcell to eliminate disease-causing cells in any given disease.

In addition, DVD-Ig can be used to trigger receptor clustering andactivation when it is designed to target two different epitopes on thesame receptor. This may have benefit in making agonistic andantagonistic anti-GPCR therapeutics. In this case, DVD-Ig can be used totarget two different epitopes on one cell for clustering/signaling (twocell surface molecules) or signaling (on one molecule). Similarly, aDVD-Ig molecule can be designed to trigger CTLA-4 ligation, and anegative signal by targeting two different epitopes (or 2 copies of thesame epitope) of CTLA-4 extracellular domain, leading to down regulationof the immune response. CTLA-4 is a clinically validated target fortherapeutic treatment of a number of immunological disorders. CTLA-4/B7interactions negatively regulate T cell activation by attenuating cellcycle progression, IL-2 production, and proliferation of T cellsfollowing activation, and CTLA-4 (CD152) engagement can down-regulate Tcell activation and promote the induction of immune tolerance. However,the strategy of attenuating T cell activation by agonistic antibodyengagement of CTLA-4 has been unsuccessful since CTLA-4 activationrequires ligation. The molecular interaction of CTLA-4/B7 is in “skewedzipper” arrays, as demonstrated by crystal structural analysis (Stamper2001 Nature 410:608). However none of the currently available CTLA-4binding reagents have ligation properties, including anti-CTLA-4monoclonal antibodies. There have been several attempts to address thisissue. In one case, a cell member-bound single chain antibody wasgenerated, and significantly inhibited allogeneic rejection in mice(Hwang 2002 JI 169:633). In a separate case, artificial APCsurface-linked single-chain antibody to CTLA-4 was generated anddemonstrated to attenuate T cell responses (Griffin 2000 JI 164:4433).In both cases, CTLA-4 ligation was achieved by closely localizedmember-bound antibodies in artificial systems. While these experimentsprovide proof-of-concept for immune down-regulation by triggering CTLA-4negative signaling, the reagents used in these reports are not suitablefor therapeutic use. To this end, CTLA-4 ligation may be achieved byusing a DVD-Ig molecule, which target two different epitopes (or 2copies of the same epitope) of CTLA-4 extracellular domain. Therationale is that the distance spanning two binding sites of an IgG,approximately 150-170 Å, is too large for active ligation of CTLA-4(30-50 Å between 2 CTLA-4 homodimer). However the distance between thetwo binding sites on DVD-Ig (one arm) is much shorter, also in the rangeof 30-50 Å, allowing proper ligation of CTLA-4.

Similarly, DVD-Ig can target two different members of a cell surfacereceptor complex (e.g. IL-12R alpha and beta). Furthermore, DVD-Ig cantarget CR1 and a soluble protein/pathogen to drive rapid clearance ofthe target soluble protein/pathogen.

Additionally, DVD-Igs of the invention can be employed fortissue-specific delivery (target a tissue marker and a disease mediatorfor enhanced local PK thus higher efficacy and/or lower toxicity),including intracellular delivery (targeting an internalizing receptorand a intracellular molecule), delivering to inside brain (targetingtransferrin receptor and a CNS disease mediator for crossing theblood-brain barrier). DVD-Ig can also serve as a carrier protein todeliver an antigen to a specific location via binding to anon-neutralizing epitope of that antigen and also to increase thehalf-life of the antigen. Furthermore, DVD-Ig can be designed to eitherbe physically linked to medical devices implanted into patients ortarget these medical devices (see Burke, Sandra E.; Kuntz, Richard E.;Schwartz, Lewis B., Zotarolimus (ABT-578) eluting stents. Advanced DrugDelivery Reviews (2006), 58(3), 437-446; Surface coatings for biologicalactivation and functionalization of medical devices, Hildebrand, H. F.;Blanchemain, N.; Mayer, G.; Chai, F.; Lefebvre, M.; Boschin, F., Surfaceand Coatings Technology (2006), 200(22-23), 6318-6324; Drug/devicecombinations for local drug therapies and infection prophylaxis, Wu,Peng; Grainger, David W., Biomaterials (2006), 27(11), 2450-2467;Mediation of the cytokine network in the implantation of orthopedicdevices, Marques, A. P.; Hunt, J. A.; Reis, Rui L., BiodegradableSystems in Tissue Engineering and Regenerative Medicine (2005),377-397). Briefly, directing appropriate types of cell to the site ofmedical implant may promote healing and restoring normal tissuefunction. Alternatively, inhibition of mediators (including but notlimited to cytokines), released upon device implantation by a DVDcoupled to or target to a device is also provided. For example, Stentshave been used for years in interventional cardiology to clear blockedarteries and to improve the flow of blood to the heart muscle. However,traditional bare metal stents have been known to cause restenosis(re-narrowing of the artery in a treated area) in some patients and canlead to blood clots. Recently, an anti-CD34 antibody coated stent hasbeen described which reduced restenosis and prevents blood clots fromoccurring by capturing endothelial progenitor cells (EPC) circulatingthroughout the blood. Endothelial cells are cells that line bloodvessels, allowing blood to flow smoothly. The EPCs adhere to the hardsurface of the stent forming a smooth layer that not only promoteshealing but prevents restenosis and blood clots, complicationspreviously associated with the use of stents (Aoji et al. 2005 J Am CollCardiol. 45(10):1574-9). In addition to improving outcomes for patientsrequiring stents, there are also implications for patients requiringcardiovascular bypass surgery. For example, a prosthetic vascularconduit (artificial artery) coated with anti-EPC antibodies wouldeliminate the need to use arteries from patients legs or arms for bypasssurgery grafts. This would reduce surgery and anesthesia times, which inturn will reduce coronary surgery deaths. DVD-Ig are designed in such away that it binds to a cell surface marker (such as CD34) as well as aprotein (or an epitope of any kind, including but not limited to lipidsand polysaccharides) that has been coated on the implanted device tofacilitate the cell recruitment. Such approaches can also be applied toother medical implants in general. Alternatively, DVD-Igs can be coatedon medical devices and upon implantation and releasing all DVDs from thedevice (or any other need which may require additional fresh DVD-Ig,including aging and denaturation of the already loaded DVD-Ig) thedevice could be reloaded by systemic administration of fresh DVD-Ig tothe patient, where the DVD-Ig is designed to binds to a target ofinterest (a cytokine, a cell surface marker (such as CD34) etc.) withone set of binding sites and to a target coated on the device (includinga protein, an epitope of any kind, including but not limited to lipids,polysaccharides and polymers) with the other. This technology has theadvantage of extending the usefulness of coated implants.

A. Use of DVD-Igs in Various Diseases

DVD-Ig molecules of the invention are also useful as therapeuticmolecules to treat various diseases. Such DVD molecules may bind one ormore targets involved in a specific disease. Examples of such targets invarious diseases are described below.

1. Human Autoimmune and Inflammatory Response

Many proteins have been implicated in general autoimmune andinflammatory responses, including C5, CCL1 (1-309), CCL11 (eotaxin),CCL13 (mcp-4), CCL15 (MIP-1d), CCL16 (HCC-4), CCL17 (TARC), CCL18(PARC), CCL19, CCL2 (mcp-1), CCL20 (MIP-3a), CCL21 (MIP-2), CCL23(MPIF-1), CCL24 (MPIF-2/eotaxin-2), CCL25 (TECK), CCL26, CCL3 (MIP-1a),CCL4 (MIP-1b), CCL5 (RANTES), CCL7 (mcp-3), CCL8 (mcp-2), CXCL1, CXCL10(IP-10), CXCL11 (1-TAC/IP-9), CXCL12 (SDF1), CXCL13, CXCL14, CXCL2,CXCL3, CXCL5 (ENA-78/LIX), CXCL6 (GCP-2), CXCL9, IL13, IL8, CCL13(mcp-4), CCR1, CCR2, CCR3, CCR4, CCR5, CCR6, CCR7, CCR8, CCR9, CX3CR1,IL8RA, XCR1 (CCXCR1), IFNA2, IL10, IL13, IL17C, IL1A, IL1B, IL1F10,IL1F5, IL1F6, IL1F7, IL1F8, IL1F9, IL22, IL5, IL8, IL9, LTA, LTB, MIF,SCYE1 (endothelial Monocyte-activating cytokine), SPP1, TNF, TNFSF5,IFNA2, IL10RA, IL10RB, IL13, IL13RA1, IL5RA, IL9, IL9R, ABCF1, BCL6, C3,C4A, CEBPB, CRP, ICEBERG, IL1R1, IL1RN, IL8RB, LTB4R, TOLLIP, FADD,IRAK1, IRAK2, MYD88, NCK2, TNFAIP3, TRADD, TRAF1, TRAF2, TRAF3, TRAF4,TRAF5, TRAF6, ACVR1, ACVR1B, ACVR2, ACVR2B, ACVRL1, CD28, CD3E, CD3G,CD3Z, CD69, CD80, CD86, CNR1, CTLA4, CYSLTR1, FCER1A, FCER2, FCGR3A,GPR44, HAVCR2, OPRD1, P2Rx7, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8,TLR9, TLR10, BLR1, CCL1, CCL2, CCL3, CCL4, CCL5, CCL7, CCL8, CCL11,CCL13, CCL15, CCL16, CCL17, CCL18, CCL19, CCL20, CCL21, CCL22, CCL23,CCL24, CCL25, CCR1, CCR2, CCR3, CCR4, CCR5, CCR6, CCR7, CCR8, CCR9,CX3CL1, CX3CR1, CXCL1, CXCL2, CXCL3, CXCL5, CXCL6, CXCL10, CXCL11,CXCL12, CXCL13, CXCR4, GPR2, SCYE1, SDF2, XCL1, XCL2, XCR1, AMH, AMHR2,BMPR1A, BMPR1B, BMPR2, C19orf10 (IL27w), CER1, CSF1, CSF2, CSF3,DKFZp451J0118, FGF2, GFI1, IFNA1, IFNB1, IFNG, IGF1, IL1A, IL1B, IL1R1,IL1R2, IL2, IL2RA, IL2RB, IL2RG, IL3, IL4, IL4R, IL5, IL5RA, IL6, IL6R,IL6ST, IL7, IL8, IL8RA, IL8RB, IL9, IL9R, IL10, IL10RA, IL10RB, IL11,IL11RA, IL12A, IL12B, IL12RB1, IL12RB2, IL13, IL13RA1, IL13RA2, IL15,IL15RA, IL16, IL17, IL17R, IL18, IL18R1, IL19, IL20, KITLG, LEP, LTA,LTB, LTB4R, LTB4R2, LTBR, MIF, NPPB, PDGFB, TBX21, TDGF1, TGFA, TGFB1,TGFB11, TGFB2, TGFB3, TGFB1, TGFBR1, TGFBR2, TGFBR3, TH1L, TNF,TNFRSF1A, TNFRSF1B, TNFRSF7, TNFRSF8, TNFRSF9, TNFRSF11A, TNFRSF21,TNFSF4, TNFSF5, TNFSF6, TNFSF11, VEGF, ZFPM2, and RNF110 (ZNF144). Inone aspect, DVD-Igs capable of binding one or more of the targets listedabove are provided.

2. Asthma

Allergic asthma is characterized by the presence of eosinophilia, gobletcell metaplasia, epithelial cell alterations, airway hyperreactivity(AHR), and Th2 and Th1 cytokine expression, as well as elevated serumIgE levels. It is now widely accepted that airway inflammation is thekey factor underlying the pathogenesis of asthma, involving a complexinterplay of inflammatory cells such as T cells, B cells, eosinophils,mast cells and macrophages, and of their secreted mediators includingcytokines and chemokines. Corticosteroids are the most importantanti-inflammatory treatment for asthma today, however their mechanism ofaction is non-specific and safety concerns exist, especially in thejuvenile patient population. The development of more specific andtargeted therapies is therefore warranted. There is increasing evidencethat IL-13 in mice mimics many of the features of asthma, including AHR,mucus hypersecretion and airway fibrosis, independently of eosinophilicinflammation (Finotto et al., International Immunology (2005), 17(8),993-1007; Padilla et al., Journal of Immunology (2005), 174(12),8097-8105).

IL-13 has been implicated as having a pivotal role in causingpathological responses associated with asthma. The development ofanti-IL-13 monoclonal antibody therapy to reduce the effects of IL-13 inthe lung is an exciting new approach that offers considerable promise asa novel treatment for asthma. However other mediators of differentialimmunological pathways are also involved in asthma pathogenesis, andblocking these mediators, in addition to IL-13, may offer additionaltherapeutic benefit. Such target pairs include, but are not limited to,IL-13 and a pro-inflammatory cytokine, such as tumor necrosis factor-α(TNF-α). TNF-α may amplify the inflammatory response in asthma and maybe linked to disease severity (McDonnell, et al., Progress inRespiratory Research (2001), 31 (New Drugs for Asthma, Allergy andCOPD), 247-250.). This suggests that blocking both IL-13 and TNF-α mayhave beneficial effects, particularly in severe airway disease. In apreferred embodiment the DVD-Ig of the invention binds the targets IL-13and TNFα and is used for treating asthma.

Animal models such as OVA-induced asthma mouse model, where bothinflammation and AHR can be assessed, are known in the art and may beused to determine the ability of various DVD-Ig molecules to treatasthma. Animal models for studying asthma are disclosed in Coffman, etal., Journal of Experimental Medicine (2005), 201(12), 1875-1879; Lloyd,et al., Advances in Immunology (2001), 77, 263-295; Boyce et al.,Journal of Experimental Medicine (2005), 201(12), 1869-1873; andSnibson, et al., Journal of the British Society for Allergy and ClinicalImmunology (2005), 35(2), 146-52. In addition to routine safetyassessments of these target pairs specific tests for the degree ofimmunosuppression may be warranted and helpful in selecting the besttarget pairs (see Luster et al., Toxicology (1994), 92(1-3), 229-43;Descotes, et al., Developments in biological standardization (1992), 7799-102; Hart et al., Journal of Allergy and Clinical Immunology (2001),108(2), 250-257).

Based on the rationale disclosed above and using the same evaluationmodel for efficacy and safety other pairs of targets that DVD-Igmolecules can bind and be useful to treat asthma may be determined.Preferably such targets include, but are not limited to, IL-13 and IL-1beta, since IL-1beta is also implicated in inflammatory response inasthma; IL-13 and cytokines and chemokines that are involved ininflammation, such as IL-13 and IL-9; IL-13 and IL-4; IL-13 and IL-5;IL-13 and IL-25; IL-13 and TARC; IL-13 and MDC; IL-13 and MIF; IL-13 andTGF-β; IL-13 and LHR agonist; IL-13 and CL25; IL-13 and SPRR2a; IL-13and SPRR2b; and IL-13 and ADAM8. The present invention also providesDVD-Igs capable of binding one or more targets involved in asthmaselected from the group consisting of CSF1 (MCSF), CSF2 (GM-CSF), CSF3(GCSF), FGF2, IFNA1, IFNB1, IFNG, histamine and histamine receptors,IL1A, IL1B, IL2, IL3, IL4, IL5, IL6, IL7, IL8, IL9, IL10, IL11, IL12A,IL12B, IL13, IL14, IL15, IL16, IL17, IL18, IL19, KITLG, PDGFB, IL2RA,IL4R, IL5RA, IL8RA, IL8RB, IL12RB1, IL12RB2, IL13RA1, IL13RA2, IL18R1,TSLP, CCL1, CCL2, CCL3, CCL4, CCL5, CCL7, CCL8, CCL13, CCL17, CCL18,CCL19, CCL20, CCL22, CCL24, CX3CL1, CXCL1, CXCL2, CXCL3, XCL1, CCR2,CCR3, CCR4, CCR5, CCR6, CCR7, CCR8, CX3CR1, GPR2, XCR1, FOS, GATA3,JAK1, JAK3, STAT6, TBX21, TGFB1, TNFSF6, YY1, CYSLTR1, FCER1A, FCER2,LTB4R, TB4R2, LTBR, and Chitinase.

3. Rheumatoid Arthritis

Rheumatoid arthritis (RA), a systemic disease, is characterized by achronic inflammatory reaction in the synovium of joints and isassociated with degeneration of cartilage and erosion of juxta-articularbone. Many pro-inflammatory cytokines including TNF, chemokines, andgrowth factors are expressed in diseased joints. Systemic administrationof anti-TNF antibody or sTNFR fusion protein to mouse models of RA wasshown to be anti-inflammatory and joint protective. Clinicalinvestigations in which the activcity of TNF in RA patients was blockedwith intravenously administered infliximab (Harriman G, Harper L K,Schaible T F. 1999 Summary of clinical trials in rheumatoid arthritisusing infliximab, an anti-TNFalpha treatment. Ann Rheum Dis 58 Suppl1:161-4.), a chimeric anti-TNF monoclonal antibody (mAB), has providedevidence that TNF regulates IL-6, IL-8, MCP-1, and VEGF production,recruitment of immune and inflammatory cells into joints, angiogenesis,and reduction of blood levels of matrix metalloproteinases-1 and -3. Abetter understanding of the inflammatory pathway in rheumatoid arthritishas led to identification of other therapeutic targets involved inrheumatoid arthritis. Promising treatments such as interleukin-6antagonists (MRA), CTLA4Ig (abatacept, Genovese Mc et al 2005 Abataceptfor rheumatoid arthritis refractory to tumor necrosis factor alphainhibition. N Engl J Med. 353:1114-23.), and anti-B cell therapy(rituximab, Okamoto H, Kamatani N. 2004 Rituximab for rheumatoidarthritis. N Engl J Med. 351:1909) have already been tested inrandomized controlled trials over the past year. Other cytokines havebeen identified and have been shown to be of benefit in animal models,including interleukin-15, interleukin-17, and interleukin-18, andclinical trials of these agents are currently under way. Dual-specificantibody therapy, combining anti-TNF and another mediator, has greatpotential in enhancing clinical efficacy and/or patient coverage. Forexample, blocking both TNF and VEGF can potentially eradicateinflammation and angiogenesis, both of which are involved inpathophysiology of RA. Blocking other pairs of targets involved in RAincluding, but not limited to, TNF and IL-18; TNF and IL-12; TNF andIL-23; TNF and IL-1beta; TNF and MIF; TNF and IL-17; and TNF and IL-15with specific DVD Igs is also contemplated. In addition to routinesafety assessments of these target pairs, specific tests for the degreeof immunosuppression may be warranted and helpful in selecting the besttarget pairs (see Luster et al., Toxicology (1994), 92(1-3), 229-43;Descotes, et al., Developments in biological standardization (1992), 7799-102; Hart et al., Journal of Allergy and Clinical Immunology (2001),108(2), 250-257). Whether a DVD Ig molecule will be useful for thetreatment of rheumatoid arthritis can be assessed using pre-clinicalanimal RA models such as the collagen-induced arthritis mouse model.Other useful models are also well known in the art (see Brand D D., CompMed. (2005) 55(2): 114-22).

4. SLE

The immunopathogenic hallmark of SLE is the polyclonal B cellactivation, which leads to hyperglobulinemia, autoantibody productionand immune complex formation. The fundamental abnormality appears to bethe failure of T cells to suppress the forbidden B cell clones due togeneralized T cell dysregulation. In addition, B and T-cell interactionis facilitated by several cytokines such as IL-10 as well asco-stimulatory molecules such as CD40 and CD40L, B7 and CD28 and CTLA-4,which initiate the second signal. These interactions together withimpaired phagocytic clearance of immune complexes and apoptoticmaterial, perpetuate the immune response with resultant tissue injury.The following targets may be involved in SLE and can potentially be usedfor DVD-Ig approach for therapeutic intervention: B cell targetedtherapies: CD-20, CD-22, CD-19, CD28, CD4, CD80, HLA-DRA, IL10, IL2,IL4, TNFRSF5, TNFRSF6, TNFSF5, TNFSF6, BLR1, HDAC4, HDAC5, HDAC7A,HDAC9, ICOSL, IGBP1, MS4A1, RGS1, SLA2, CD81, IFNB1, IL10, TNFRSF5,TNFRSF7, TNFSF5, AICDA, BLNK, GALNAC4S-6ST, HDAC4, HDAC5, HDAC7A, HDAC9,IL10, IL11, IL4, INHA, INHBA, KLF6, TNFRSF7, CD28, CD38, CD69, CD80,CD83, CD86, DPP4, FCER2, IL2RA, TNFRSF8, TNFSF7, CD24, CD37, CD40, CD72,CD74, CD79A, CD79B, CR2, IL1R2, ITGA2, ITGA3, MS4A1, ST6GAL1, CD1C,CHST10, HLA-A, HLA-DRA, and NT5E.; co-stimulatory signals: CTLA4 orB7.1/B7.2; inhibition of B cell survival: BlyS, BAFF; Complementinactivation: C5; Cytokine modulation: the key principle is that the netbiologic response in any tissue is the result of a balance between locallevels of proinflammatory or anti-inflammatory cytokines (see Sfikakis PP et al 2005 Curr Opin Rheumatol 17:550-7). SLE is considered to be aTh-2 driven disease with documented elevations in serum IL-4, IL-6,IL-10. DVD Igs capable of binding one or more targets selected from thegroup consisting of IL-4, IL-6, IL-10, IFN-a, and TNF-a are alsocontemplated. Combination of targets discussed above will enhancetherapeutic efficacy for SLE which can be tested in a number of lupuspreclinical models (see Peng S L (2004) Methods Mol Med.; 102:227-72).

5. Multiple Sclerosis

Multiple sclerosis (MS) is a complex human autoimmune-type disease witha predominantly unknown etiology. Immunologic destruction of myelinbasic protein (MBP) throughout the nervous system is the major pathologyof multiple sclerosis. MS is a disease of complex pathologies, whichinvolves infiltration by CD4+ and CD8+ T cells and of response withinthe central nervous system. Expression in the CNS of cytokines, reactivenitrogen species and costimulator molecules have all been described inMS. Of major consideration are immunological mechanisms that contributeto the development of autoimmunity. In particular, antigen expression,cytokine and leukocyte interactions, and regulatory T-cells, which helpbalance/modulate other T-cells such as Th1 and Th2 cells, are importantareas for therapeutic target identification.

IL-12 is a proinflammatory cytokine that is produced by APC and promotesdifferentiation of Th1 effector cells. IL-12 is produced in thedeveloping lesions of patients with MS as well as in EAE-affectedanimals. Previously it was shown that interference in IL-12 pathwayseffectively prevents EAE in rodents, and that in vivo neutralization ofIL-12p40 using a anti-IL-12 mAb has beneficial effects in themyelin-induced EAE model in common marmosets.

TWEAK is a member of the TNF family, constitutively expressed in thecentral nervous system (CNS), with pro-inflammatory, proliferative orapoptotic effects depending upon cell types. Its receptor, Fn14, isexpressed in CNS by endothelial cells, reactive astrocytes and neurons.TWEAK and Fn14 mRNA expression increased in spinal cord duringexperimental autoimmune encephalomyelitis (EAE). Anti-TWEAK antibodytreatment in myelin oligodendrocyte glycoprotein (MOG) induced EAE inC57BL/6 mice resulted in a reduction of disease severity and leukocyteinfiltration when mice were treated after the priming phase.

One aspect of the invention pertains to DVD Ig molecules capable ofbinding one or more, preferably two, targets selected from the groupconsisting of IL-12, TWEAK, IL-23, CXCL13, CD40, CD40L, IL-18, VEGF,VLA-4, TNF, CD45RB, CD200, IFNgamma, GM-CSF, FGF, C5, CD52, and CCR2. Apreferred embodiment includes a dual-specific anti-IL-12/TWEAK DVD Ig asa therapeutic agent beneficial for the treatment of MS. Several animalmodels for assessing the usefulness of the DVD molecules to treat MS areknown in the art (see Steinman L, et al., (2005) Trends Immunol.26(11):565-71; Lublin F D., et al., (1985) Springer Semin Immunopathol.8(3):197-208; Genain C P, et al., (1997) J Mol Med. 75(3):187-97; TuohyV K, et al., (1999) J Exp Med. 189(7):1033-42; Owens T, et al., (1995)Neurol Clin.13(1):51-73; and 't Hart B A, et al., (2005) J Immunol175(7):4761-8. In addition to routine safety assessments of these targetpairs specific tests for the degree of immunosuppression may bewarranted and helpful in selecting the best target pairs (see Luster etal., Toxicology (1994), 92(1-3), 229-43; Descotes, et al., Developmentsin biological standardization (1992), 77 99-102; Jones R. 2000Rovelizumab (ICOS Corp). IDrugs.3(4):442-6).

6. Sepsis

The pathophysiology of sepsis is initiated by the outer membranecomponents of both gram-negative organisms (lipopolysaccharide [LPS],lipid A, endotoxin) and gram-positive organisms (lipoteichoic acid,peptidoglycan). These outer membrane components are able to bind to theCD14 receptor on the surface of monocytes. By virtue of the recentlydescribed toll-like receptors, a signal is then transmitted to the cell,leading to the eventual production of the proinflammatory cytokinestumor necrosis factor-alpha (TNF-alpha) and interleukin-1 (IL-1).Overwhelming inflammatory and immune responses are essential features ofseptic shock and play a central part in the pathogenesis of tissuedamage, multiple organ failure, and death induced by sepsis. Cytokines,especially tumor necrosis factor (TNF) and interleukin (IL)-1, have beenshown to be critical mediators of septic shock. These cytokines have adirect toxic effect on tissues; they also activate phospholipase A2.These and other effects lead to increased concentrations ofplatelet-activating factor, promotion of nitric oxide synthase activity,promotion of tissue infiltration by neutrophils, and promotion ofneutrophil activity.

The treatment of sepsis and septic shock remains a clinical conundrum,and recent prospective trials with biological response modifiers (i.e.anti-TNF, anti-MIF) aimed at the inflammatory response have shown onlymodest clinical benefit. Recently, interest has shifted toward therapiesaimed at reversing the accompanying periods of immune suppression.Studies in experimental animals and critically ill patients havedemonstrated that increased apoptosis of lymphoid organs and someparenchymal tissues contribute to this immune suppression, anergy, andorgan system dysfunction. During sepsis syndromes, lymphocyte apoptosiscan be triggered by the absence of IL-2 or by the release ofglucocorticoids, granzymes, or the so-called ‘death’ cytokines: tumornecrosis factor alpha or Fas ligand. Apoptosis proceeds viaauto-activation of cytosolic and/or mitochondrial caspases, which can beinfluenced by the pro- and anti-apoptotic members of the Bcl-2 family.In experimental animals, not only can treatment with inhibitors ofapoptosis prevent lymphoid cell apoptosis; it may also improve outcome.Although clinical trials with anti-apoptotic agents remain distant duein large part to technical difficulties associated with theiradministration and tissue targeting, inhibition of lymphocyte apoptosisrepresents an attractive therapeutic target for the septic patient.Likewise, a dual-specific agent targeting both inflammatory mediator anda apoptotic mediator, may have added benefit. One aspect of theinvention pertains to DVD Igs capable of binding one or more targetsinvolved in sepsis, preferably two targets, selected from the groupconsisting TNF, IL-1, MIF, IL-6, IL-8, IL-18, IL-12, IL-23, FasL, LPS,Toll-like receptors, TLR-4, tissue factor, MIP-2, ADORA2A, CASP1, CASP4,IL10, IL1B, NFKB1, PROC, TNFRSF1A, CSF3, IL10, IL1B, IL6, ADORA2A, CCR3,IL10, IL1B, IL1RN, MIF, NFKB1, PTAFR, TLR2, TLR4, GPR44, HMOX1, midkine,IRAK1, NFKB2, SERPINA1, SERPINE1, and TREM1. The efficacy of such DVDIgs for sepsis can be assessed in preclinical animal models known in theart (see Buras J A, et al., (2005) Nat Rev Drug Discov. 4(10):854-65 andCalandra T, et al., (2000) Nat Med. 6(2): 164-70).

7. Neurological Disorders

7.1. Neurodegenerative Diseases

Chronic neurodegenerative diseases are usually age-dependent diseasescharacterized by progressive loss of neuronal functions (neuronal celldeath, demyelination), loss of mobility and loss of memory. Emergingknowledge of the mechanisms underlying chronic neurodegenerativediseases (e.g. Alzheimer's disease) show a complex etiology and avariety of factors have been recognized to contribute to theirdevelopment and progression e.g. age, glycemic status, amyloidproduction and multimerization, accumulation of advanced glycation-endproducts (AGE) which bind to their receptor RAGE (receptor for AGE),increased brain oxidative stress, decreased cerebral blood flow,neuroinflammation including release of inflammatory cytokines andchemokines, neuronal dysfunction and microglial activation. Thus thesechronic neurodegenerative diseases represent a complex interactionbetween multiple cell types and mediators. Treatment strategies for suchdiseases are limited and mostly constitute either blocking inflammatoryprocesses with non-specific anti-inflammatory agents (egcorticosteroids, COX inhibitors) or agents to prevent neuron loss and/orsynaptic functions. These treatments fail to stop disease progression.Recent studies suggest that more targeted therapies such as antibodiesto soluble A-b peptide (including the A-b oligomeric forms) can not onlyhelp stop disease progression but may help maintain memory as well.These preliminary observations suggest that specific therapies targetingmore than one disease mediator (e.g. A-b and a pro-inflammatory cytokinesuch as TNF) may provide even better therapeutic efficacy for chronicneurodegenerative diseases than observed with targeting a single diseasemechanism (e.g. soluble A-balone) (see C. E. Shepherd, et al, NeurobiolAging. 2005 Oct. 24; Nelson R B., Curr Pharm Des. 2005; 11:3335; WilliamL. Klein.; Neurochem Int. 2002; 41: 345; Michelle C Janelsins, et al., JNeuroinflammation. 2005; 2: 23; Soloman B., Curr Alzheimer Res. 2004; 1:149; Igor Klyubin, et al., Nat Med. 2005; 11: 556-61; Arancio O, et al.,EMBO Journal (2004) 1-10; Bomemann K D, et al., Am J Pathol. 2001; 158:63; Deane R, et al., Nat Med. 2003; 9: 907-13; and Eliezer Masliah, etal., Neuron. 2005; 46: 857).

The DVD-Ig molecules of the invention can bind one or more targetsinvolved in Chronic neurodegenerative diseases such as Alzheimers. Suchtargets include, but are not limited to, any mediator, soluble or cellsurface, implicated in AD pathogenesis e.g AGE (S100 A, amphoterin),pro-inflammatory cytokines (e.g. IL-1), chemokines (e.g. MCP 1),molecules that inhibit nerve regeneration (e.g. Nogo, RGM A), moleculesthat enhance neurite growth (neurotrophins). The efficacy of DVD-Igmolecules can be validated in pre-clinical animal models such as thetransgenic mice that over-express amyloid precursor protein or RAGE anddevelop Alzheimer's disease-like symptoms. In addition, DVD-Ig moleculescan be constructed and tested for efficacy in the animal models and thebest therapeutic DVD-Ig can be selected for testing in human patients.DVD-Ig molecules can also be employed for treatment of otherneurodegenerative diseases such as Parkinson's disease. Alpha-Synucleinis involved in Parkinson's pathology. A DVD-Ig capable of targetingalpha-synuclein and inflammatory mediators such as TNF, IL-1, MCP-1 canprove effective therapy for Parkinson's disease and are contemplated inthe invention.

7.2 Neuronal Regeneration and Spinal Cord Injury

Despite an increase in knowledge of the pathologic mechanisms, spinalcord injury (SCI) is still a devastating condition and represents amedical indication characterized by a high medical need. Most spinalcord injuries are contusion or compression injuries and the primaryinjury is usually followed by secondary injury mechanisms (inflammatorymediators e.g. cytokines and chemokines) that worsen the initial injuryand result in significant enlargement of the lesion area, sometimes morethan 10-fold. These primary and secondary mechanisms in SCI are verysimilar to those in brain injury caused by other means e.g. stroke. Nosatisfying treatment exists and high dose bolus injection ofmethylprednisolone (MP) is the only used therapy within a narrow timewindow of 8 h post injury. This treatment, however, is only intended toprevent secondary injury without causing any significant functionalrecovery. It is heavily critisized for the lack of unequivocal efficacyand severe adverse effects, like immunosuppression with subsequentinfections and severe histopathological muscle alterations. No otherdrugs, biologics or small molecules, stimulating the endogenousregenerative potential are approved, but promising treatment principlesand drug candidates have shown efficacy in animal models of SCI inrecent years. To a large extent the lack of functional recovery in humanSCI is caused by factors inhibiting neurite growth, at lesion sites, inscar tissue, in myelin as well as on injury-associated cells. Suchfactors are the myelin-associated proteins NogoA, OMgp and MAG, RGM A,the scar-associated CSPG (Chondroitin Sulfate Proteoglycans) andinhibitory factors on reactive astrocytes (some semaphorins andephrins). However, at the lesion site not only growth inhibitorymolecules are found but also neurite growth stimulating factors likeneurotrophins, laminin, L1 and others. This ensemble of neurite growthinhibitory and growth promoting molecules may explain that blockingsingle factors, like NogoA or RGM A, resulted in significant functionalrecovery in rodent SCI models, because a reduction of the inhibitoryinfluences could shift the balance from growth inhibition to growthpromotion. However, recoveries observed with blocking a single neuriteoutgrowth inhibitory molecule were not complete. To achieve faster andmore pronounced recoveries either blocking two neurite outgrowthinhibitory molecules e.g Nogo and RGM A, or blocking an neuriteoutgrowth inhibitory molecule and enhancing functions of a neuriteoutgrowth enhancing molecule e.g Nogo and neurotrophins, or blocking aneurite outgrowth inhibitory moleclule e.g. Nogo and a pro-inflammatorymolecule e.g. TNF, may be desirable (see McGee A W, et al., TrendsNeurosci. 2003; 26: 193; Marco Domeniconi, et al., J Neurol Sci. 2005;233:43; Milan Makwanal, et al., FEBS J. 2005; 272:2628; Barry J.Dickson, Science. 2002; 298: 1959; Felicia Yu Hsuan Teng, et al., JNeurosci Res. 2005; 79:273; Tara Karnezis, et al., Nature Neuroscience2004; 7, 736; Gang Xu, et al., J. Neurochem. 2004; 91; 1018).

In one aspect, DVD-Igs capable of binding target pairs such as NgR andRGM A; NogoA and RGM A; MAG and RGM A; OMGp and RGM A; RGM A and RGM B;CSPGs and RGM A; aggrecan, midkine, neurocan, versican, phosphacan, Te38and TNF-a; Aβ globulomer-specific antibodies combined with antibodiespromoting dendrite & axon sprouting are provided. Dendrite pathology isa very early sign of AD and it is known that NOGO A restricts dendritegrowth. One can combine such type of ab with any of the SCI-candidate(myelin-proteins) Ab. Other DVD-Ig targets may include any combinationof NgR-p75, NgR-Troy, NgR-Nogo66 (Nogo), NgR-Lingo, Lingo-Troy,Lingo-p75, MAG or Omgp. Additionally, targets may also include anymediator, soluble or cell surface, implicated in inhibition of neuritee.g Nogo, Ompg, MAG, RGM A, semaphorins, ephrins, soluble A-b,pro-inflammatory cytokines (e.g. IL-1), chemokines (e.g. MIP 1a),molecules that inhibit nerve regeneration. The efficacy ofanti-nogo/anti-RGM A or similar DVD-Ig molecules can be validated inpre-clinical animal models of spinal cord injury. In addition, theseDVD-Ig molecules can be constructed and tested for efficacy in theanimal models and the best therapeutic DVD-Ig can be selected fortesting in human patients. In addition, DVD-Ig molecules can beconstructed that target two distinct ligand binding sites on a singlereceptor e.g. Nogo receptor which binds three ligand Nogo, Ompg, and MAGand RAGE that binds A-b and S100 A. Furthermore, neurite outgrowthinhibitors e.g. nogo and nogo receptor, also play a role in preventingnerve regeneration in immunological diseases like multiple sclerosis.Inhibition of nogo-nogo receptor interaction has been shown to enhancerecovery in animal models of multiple sclerosis. Therefore, DVD-Igmolecules that can block the function of one immune mediator eg acytokine like IL-12 and a neurite outgrowth inhibitor molecule eg nogoor RGM may offer faster and greater efficacy than blocking either animmune or an neurite outgrowth inhibitor molecule alone.

8. Oncological Disorders

Monoclonal antibody therapy has emerged as an important therapeuticmodality for cancer (von Mehren M, et al 2003 Monoclonal antibodytherapy for cancer. Annu Rev Med.; 54: 343-69). Antibodies may exertantitumor effects by inducing apoptosis, redirected cytotoxicity,interfering with ligand-receptor interactions, or preventing theexpression of proteins that are critical to the neoplastic phenotype. Inaddition, antibodies can target components of the tumormicroenvironment, perturbing vital structures such as the formation oftumor-associated vasculature. Antibodies can also target receptors whoseligands are growth factors, such as the epidermal growth factorreceptor. The antibody thus inhibits natural ligands that stimulate cellgrowth from binding to targeted tumor cells. Alternatively, antibodiesmay induce an anti-idiotype network, complement-mediated cytotoxicity,or antibody-dependent cellular cytotoxicity (ADCC). The use ofdual-specific antibody that targets two separate tumor mediators willlikely give additional benefit compared to a mono-specific therapy. DVDIgs capable of binding the following pairs of targets to treatoncological disease are also contemplated: IGF1 and IGF2; IGF1/2 andErb2B; VEGFR and EGFR; CD20 and CD3, CD138 and CD20, CD38 and CD20, CD38& CD138, CD40 and CD20, CD138 and CD40, CD38 and CD40. Other targetcombinations include one or more members of the EGF/erb-2/erb-3 family.Other targets (one or more) involved in oncological diseases that DVDIgs may bind include, but are not limited to those selected from thegroup consisting of: CD52, CD20, CD19, CD3, CD4, CD8, BMP6, IL12A, IL1A,IL1B, IL2, IL24, INHA, TNF, TNFSF10, BMP6, EGF, FGF1, FGF10, FGF11,FGF12, FGF13, FGF14, FGF16, FGF17, FGF18, FGF19, FGF2, FGF20, FGF21,FGF22, FGF23, FGF3, FGF4, FGF5, FGF6, FGF7, FGF8, FGF9, GRP, IGF1, IGF2,IL12A, IL1A, IL1B, IL2, INHA, TGFA, TGFB1, TGFB2, TGFB3, VEGF, CDK2,EGF, FGF10, FGF18, FGF2, FGF4, FGF7, IGF1, IGF1R, IL2, VEGF, BCL2,CD164, CDKN1A, CDKN1B, CDKN1C, CDKN2A, CDKN2B, CDKN2C, CDKN3, GNRH1,IGFBP6, IL1A, IL1B, ODZ1, PAWR, PLG, TGFB11, AR, BRCA1, CDK3, CDK4,CDK5, CDK6, CDK7, CDK9, E2F1, EGFR, ENO1, ERBB2, ESR1, ESR2, IGFBP3,IGFBP6, IL2, INSL4, MYC, NOX5, NR6A1, PAP, PCNA, PRKCQ, PRKD1, PRL,TP53, FGF22, FGF23, FGF9, IGFBP3, IL2, INHA, KLK6, TP53, CHGB, GNRH1,IGF1, IGF2, INHA, INSL3, INSL4, PRL, KLK6, SHBG, NR1D1, NR1H3, NR1I3,NR2F6, NR4A3, ESR1, ESR2, NR0B1, NR0B2, NR1D2, NR1H2, NR1H4, NR1I2,NR2C1, NR2C2, NR2E1, NR2E3, NR2F1, NR2F2, NR3C1, NR3C2, NR4A1, NR4A2,NR5A1, NR5A2, NR6A1, PGR, RARB, FGF1, FGF2, FGF6, KLK3, KRT1, APOC1,BRCA1, CHGA, CHGB, CLU, COL1A1, COL6A1, EGF, ERBB2, ERK8, FGF1, FGF10,FGF11, FGF13, FGF14, FGF16, FGF17, FGF18, FGF2, FGF20, FGF21, FGF22,FGF23, FGF3, FGF4, FGF5, FGF6, FGF7, FGF8, FGF9, GNRH1, IGF1, IGF2,IGFBP3, IGFBP6, IL12A, IL1A, IL1B, IL2, IL24, INHA, INSL3, INSL4, KLK10,KLK12, KLK13, KLK14, KLK15, KLK3, KLK4, KLK5, KLK6, KLK9, MMP2, MMP9,MSMB, NTN4, ODZ1, PAP, PLAU, PRL, PSAP, SERPINA3, SHBG, TGFA, TIMP3,CD44, CDH1, CDH10, CDH19, CDH20, CDH7, CDH9, CDH1, CDH10, CDH13, CDH18,CDH19, CDH20, CDH7, CDH8, CDH9, ROBO2, CD44, ILK, ITGA1, APC, CD164,COL6A1, MTSS1, PAP, TGFB11, AGR2, AIG1, AKAP1, AKAP2, CANT1, CAV1,CDH12, CLDN3, CLN3, CYB5, CYC1, DAB2IP, DES, DNCL1, ELAC2, ENO2, ENO3,FASN, FLJ12584, FLJ25530, GAGEB1, GAGEC1, GGT1, GSTP1, HIP1, HUMCYT2A,IL29, K6HF, KAI1, KRT2A, MIB1, PART1, PATE, PCA3, PIAS2, PIK3CG, PPID,PR1, PSCA, SLC2A2, SLC33A1, SLC43A1, STEAP, STEAP2, TPM1, TPM2, TRPC6,ANGPT1, ANGPT2, ANPEP, ECGF1, EREG, FGF1, FGF2, FIGF, FLT1, JAG1, KDR,LAMA5, NRP1, NRP2, PGF, PLXDC1, STAB 1, VEGF, VEGFC, ANGPTL3, BAI1,COL4A3, IL8, LAMA5, NRP1, NRP2, STAB1, ANGPTL4, PECAM1, PF4, PROK2,SERPINF1, TNFAIP2, CCL11, CCL2, CXCL1, CXCL10, CXCL3, CXCL5, CXCL6,CXCL9, IFNA1, IFNB1, IFNG, IL1B, IL6, MDK, EDG1, EFNA1, EFNA3, EFNB2,EGF, EPHB4, FGFR3, HGF, IGF1, ITGB3, PDGFA, TEK, TGFA, TGFB1, TGFB2,TGFBR1, CCL2, CDH5, COL18A1, EDG1, ENG, ITGAV, ITGB3, THBS1, THBS2, BAD,BAG1, BCL2, CCNA1, CCNA2, CCND1, CCNE1, CCNE2, CDH1 (E-cadherin), CDKN1B(p27Kip1), CDKN2A (p16INK4a), COL6A1, CTNNB1 (b-catenin), CTSB(cathepsin B), ERBB2 (Her-2), ESR1, ESR2, F3 (TF), FOSL1 (FRA-1), GATA3,GSN (Gelsolin), IGFBP2, IL2RA, IL6, IL6R, IL6ST (glycoprotein 130),ITGA6 (a6 integrin), JUN, KLK5, KRT19, MAP2K7 (c-Jun), MKI67 (Ki-67),NGFB (NGF), NGFR, NME1 (NM23A), PGR, PLAU (uPA), PTEN, SERPINB5(maspin), SERPINE1 (PAI-1), TGFA, THBS1 (thrombospondin-1), TIE (Tie-1),TNFRSF6 (Fas), TNFSF6 (FasL), TOP2A (topoisomerase Iia), TP53, AZGP1(zinc-a-glycoprotein), BPAG1 (plectin), CDKN1A (p21Wap1/Cip1), CLDN7(claudin-7), CLU (clusterin), ERBB2 (Her-2), FGF1, FLRT1 (fibronectin),GABRP (GABAa), GNAS1, ID2, ITGA6 (a6 integrin), ITGB4 (b 4 integrin),KLF5 (GC Box BP), KRT19 (Keratin 19), KRTHB6 (hair-specific type IIkeratin), MACMARCKS, MT3 (metallothionectin-III), MUC1 (mucin), PTGS2(COX-2), RAC2 (p21Rac2), S100A2, SCGB1D2 (lipophilin B), SCGB2A1(mammaglobin 2), SCGB2A2 (mammaglobin 1), SPRR1B (Spr1), THBS1, THBS2,THBS4, and TNFAIP2 (B94).

IV. Pharmaceutical Composition

The invention also provides pharmaceutical compositions comprising abinding protein, of the invention and a pharmaceutically acceptablecarrier. The pharmaceutical compositions comprising binding proteins ofthe invention are for use in, but not limited to, diagnosing, detecting,or monitoring a disorder, in preventing, treating, managing, orameliorating of a disorder or one or more symptoms thereof, and/or inresearch. In a specific embodiment, a composition comprises one or morebinding proteins of the invention. In another embodiment, thepharmaceutical composition comprises one or more binding proteins of theinvention and one or more prophylactic or therapeutic agents other thanbinding proteins of the invention for treating a disorder. Preferably,the prophylactic or therapeutic agents known to be useful for or havingbeen or currently being used in the prevention, treatment, management,or amelioration of a disorder or one or more symptoms thereof. Inaccordance with these embodiments, the composition may further compriseof a carrier, diluent or excipient.

The binding proteins of the invention can be incorporated intopharmaceutical compositions suitable for administration to a subject.Typically, the pharmaceutical composition comprises a binding protein ofthe invention and a pharmaceutically acceptable carrier. As used herein,“pharmaceutically acceptable carrier” includes any and all solvents,dispersion media, coatings, antibacterial and antifungal agents,isotonic and absorption delaying agents, and the like that arephysiologically compatible. Examples of pharmaceutically acceptablecarriers include one or more of water, saline, phosphate bufferedsaline, dextrose, glycerol, ethanol and the like, as well ascombinations thereof. In many cases, it will be preferable to includeisotonic agents, for example, sugars, polyalcohols such as mannitol,sorbitol, or sodium chloride in the composition. Pharmaceuticallyacceptable carriers may further comprise minor amounts of auxiliarysubstances such as wetting or emulsifying agents, preservatives orbuffers, which enhance the shelf life or effectiveness of the antibodyor antibody portion.

Various delivery systems are known and can be used to administer one ormore antibodies of the invention or the combination of one or moreantibodies of the invention and a prophylactic agent or therapeuticagent useful for preventing, managing, treating, or ameliorating adisorder or one or more symptoms thereof, e.g., encapsulation inliposomes, microparticles, microcapsules, recombinant cells capable ofexpressing the antibody or antibody fragment, receptor-mediatedendocytosis (see, e.g., Wu and Wu, J. Biol. Chem. 262: 4429-4432(1987)), construction of a nucleic acid as part of a retroviral or othervector, etc. Methods of administering a prophylactic or therapeuticagent of the invention include, but are not limited to, parenteraladministration (e.g., intradermal, intramuscular, intraperitoneal,intravenous and subcutaneous), epidurala administration, intratumoraladministration, and mucosal administration (e.g., intranasal and oralroutes). In addition, pulmonary administration can be employed, e.g., byuse of an inhaler or nebulizer, and formulation with an aerosolizingagent. See, e.g., U.S. Pat. Nos. 6,019,968, 5,985,320, 5,985,309,5,934,272, 5,874,064, 5,855,913, 5,290,540, and 4,880,078; and PCTPublication Nos. WO 92/19244, WO 97/32572, WO 97/44013, WO 98/31346, andWO 99/66903, each of which is incorporated herein by reference theirentireties. In one embodiment, a binding protein of the invention,combination therapy, or a composition of the invention is administeredusing Alkermes AIR® pulmonary drug delivery technology (Alkermes, Inc.,Cambridge, Mass.). In a specific embodiment, prophylactic or therapeuticagents of the invention are administered intramuscularly, intravenously,intratumorally, orally, intranasally, pulmonary, or subcutaneously. Theprophylactic or therapeutic agents may be administered by any convenientroute, for example by infusion or bolus injection, by absorption throughepithelial or mucocutaneous linings (e.g., oral mucosa, rectal andintestinal mucosa, etc.) and may be administered together with otherbiologically active agents. Administration can be systemic or local.

In a specific embodiment, it may be desirable to administer theprophylactic or therapeutic agents of the invention locally to the areain need of treatment; this may be achieved by, for example, and not byway of limitation, local infusion, by injection, or by means of animplant, said implant being of a porous or non-porous material,including membranes and matrices, such as sialastic membranes, polymers,fibrous matrices (e.g., TISSUEL®), or collagen matrices. In oneembodiment, an effective amount of one or more antibodies of theinvention antagonists is administered locally to the affected area to asubject to prevent, treat, manage, and/or ameliorate a disorder or asymptom thereof. In another embodiment, an effective amount of one ormore antibodies of the invention is administered locally to the affectedarea in combination with an effective amount of one or more therapies(e.g., one or more prophylactic or therapeutic agents) other than abinding protein of the invention of a subject to prevent, treat, manage,and/or ameliorate a disorder or one or more symptoms thereof.

In another embodiment, the prophylactic or therapeutic agent can bedelivered in a controlled release or sustained release system. In oneembodiment, a pump may be used to achieve controlled or sustainedrelease (see Langer, supra; Sefton, 1987, CRC Crit. Ref. Biomed. Eng.14:20; Buchwald et al., 1980, Surgery 88:507; Saudek et al., 1989, N.Engl. J. Med. 321:574). In another embodiment, polymeric materials canbe used to achieve controlled or sustained release of the therapies ofthe invention (see e.g., Medical Applications of Controlled Release,Langer and Wise (eds.), CRC Pres., Boca Raton, Fla. (1974); ControlledDrug Bioavailability, Drug Product Design and Performance, Smolen andBall (eds.), Wiley, New York (1984); Ranger and Peppas, 1983, J.Macromol. Sci. Rev. Macromol. Chem. 23:61; see also Levy et al., 1985,Science 228:190; During et al., 1989, Ann. Neurol. 25:351; Howard etal., 1989, J. Neurosurg. 7 1:105); U.S. Pat. No. 5,679,377; U.S. Pat.No. 5,916,597; U.S. Pat. No. 5,912,015; U.S. Pat. No. 5,989,463; U.S.Pat. No. 5,128,326; PCT Publication No. WO 99/15154; and PCT PublicationNo. WO 99/20253. Examples of polymers used in sustained releaseformulations include, but are not limited to, poly(2-hydroxy ethylmethacrylate), poly(methyl methacrylate), poly(acrylic acid),poly(ethylene-co-vinyl acetate), poly(methacrylic acid), polyglycolides(PLG), polyanhydrides, poly(N-vinyl pyrrolidone), poly(vinyl alcohol),polyacrylamide, poly(ethylene glycol), polylactides (PLA),poly(lactide-co-glycolides) (PLGA), and polyorthoesters. In a preferredembodiment, the polymer used in a sustained release formulation isinert, free of leachable impurities, stable on storage, sterile, andbiodegradable. In yet another embodiment, a controlled or sustainedrelease system can be placed in proximity of the prophylactic ortherapeutic target, thus requiring only a fraction of the systemic dose(see, e.g., Goodson, in Medical Applications of Controlled Release,supra, vol. 2, pp. 115-138 (1984)).

Controlled release systems are discussed in the review by Langer (1990,Science 249:1527-1533). Any technique known to one of skill in the artcan be used to produce sustained release formulations comprising one ormore therapeutic agents of the invention. See, e.g., U.S. Pat. No.4,526,938, PCT publication WO 91/05548, PCT publication WO 96/20698,Ning et al., 1996, “Intratumoral Radioimmunotheraphy of a Human ColonCancer Xenograft Using a Sustained-Release Gel,” Radiotherapy &Oncology39:179-189, Song et al., 1995, “Antibody Mediated Lung Targeting ofLong-Circulating Emulsions,” PDA Journal of Pharmaceutical Science&Technology 50:372-397, Cleek et al., 1997, “Biodegradable PolymericCarriers for a bFGF Antibody for Cardiovascular Application,” Pro.Int'l. Symp. Control. R^(e1). Bioact. Mater. 24:853-854, and Lam et al.,1997, “Microencapsulation of Recombinant Humanized Monoclonal Antibodyfor Local Delivery,” Proc. Int'l. Symp. Control Rel. Bioact. Mater.24:759-760, each of which is incorporated herein by reference in theirentireties.

In a specific embodiment, where the composition of the invention is anucleic acid encoding a prophylactic or therapeutic agent, the nucleicacid can be administered in vivo to promote expression of its encodedprophylactic or therapeutic agent, by constructing it as part of anappropriate nucleic acid expression vector and administering it so thatit becomes intracellular, e.g., by use of a retroviral vector (see U.S.Pat. No. 4,980,286), or by direct injection, or by use of microparticlebombardment (e.g., a gene gun; Biolistic, Dupont), or coating withlipids or cell-surface receptors or transfecting agents, or byadministering it in linkage to a homeobox-like peptide which is known toenter the nucleus (see, e.g., Joliot et al., 1991, Proc. Natl. Acad.Sci. USA 88:1864-1868). Alternatively, a nucleic acid can be introducedintracellularly and incorporated within host cell DNA for expression byhomologous recombination.

A pharmaceutical composition of the invention is formulated to becompatible with its intended route of administration. Examples of routesof administration include, but are not limited to, parenteral, e.g.,intravenous, intradermal, subcutaneous, oral, intranasal (e.g.,inhalation), transdermal (e.g., topical), transmucosal, and rectaladministration. In a specific embodiment, the composition is formulatedin accordance with routine procedures as a pharmaceutical compositionadapted for intravenous, subcutaneous, intramuscular, oral, intranasal,or topical administration to human beings. Typically, compositions forintravenous administration are solutions in sterile isotonic aqueousbuffer. Where necessary, the composition may also include a solubilizingagent and a local anesthetic such as lignocamne to ease pain at the siteof the injection.

If the compositions of the invention are to be administered topically,the compositions can be formulated in the form of an ointment, cream,transdermal patch, lotion, gel, shampoo, spray, aerosol, solution,emulsion, or other form well-known to one of skill in the art. See,e.g., Remington's Pharmaceutical Sciences and Introduction toPharmaceutical Dosage Forms, 19th ed., Mack Pub. Co., Easton, Pa.(1995). For non-sprayable topical dosage forms, viscous to semi-solid orsolid forms comprising a carrier or one or more excipients compatiblewith topical application and having a dynamic viscosity preferablygreater than water are typically employed. Suitable formulationsinclude, without limitation, solutions, suspensions, emulsions, creams,ointments, powders, liniments, salves, and the like, which are, ifdesired, sterilized or mixed with auxiliary agents (e.g., preservatives,stabilizers, wetting agents, buffers, or salts) for influencing variousproperties, such as, for example, osmotic pressure. Other suitabletopical dosage forms include sprayable aerosol preparations wherein theactive ingredient, preferably in combination with a solid or liquidinert carrier, is packaged in a mixture with a pressurized volatile(e.g., a gaseous propellant, such as freon) or in a squeeze bottle.Moisturizers or humectants can also be added to pharmaceuticalcompositions and dosage forms if desired. Examples of such additionalingredients are well-known in the art.

If the method of the invention comprises intranasal administration of acomposition, the composition can be formulated in an aerosol form,spray, mist or in the form of drops. In particular, prophylactic ortherapeutic agents for use according to the present invention can beconveniently delivered in the form of an aerosol spray presentation frompressurized packs or a nebuliser, with the use of a suitable propellant(e.g., dichlorodifluoromethane, trichlorofluoromethane,dichlorotetrafluoroethane, carbon dioxide or other suitable gas). In thecase of a pressurized aerosol the dosage unit may be determined byproviding a valve to deliver a metered amount. Capsules and cartridges(composed of, e.g., gelatin) for use in an inhaler or insufflator may beformulated containing a powder mix of the compound and a suitable powderbase such as lactose or starch. If the method of the invention comprisesoral administration, compositions can be formulated orally in the formof tablets, capsules, cachets, gelcaps, solutions, suspensions, and thelike. Tablets or capsules can be prepared by conventional means withpharmaceutically acceptable excipients such as binding agents (e.g.,pregelatinised maize starch, polyvinylpyrrolidone, or hydroxypropylmethylcellulose); fillers (e.g., lactose, microcrystalline cellulose, orcalcium hydrogen phosphate); lubricants (e.g., magnesium stearate, talc,or silica); disintegrants (e.g., potato starch or sodium starchglycolate); or wetting agents (e.g., sodium lauryl sulphate). Thetablets may be coated by methods well-known in the art. Liquidpreparations for oral administration may take the form of, but notlimited to, solutions, syrups or suspensions, or they may be presentedas a dry product for constitution with water or other suitable vehiclebefore use. Such liquid preparations may be prepared by conventionalmeans with pharmaceutically acceptable additives such as suspendingagents (e.g., sorbitol syrup, cellulose derivatives, or hydrogenatededible fats); emulsifying agents (e.g., lecithin or acacia); non-aqueousvehicles (e.g., almond oil, oily esters, ethyl alcohol, or fractionatedvegetable oils); and preservatives (e.g., methyl orpropyl-p-hydroxybenzoates or sorbic acid). The preparations may alsocontain buffer salts, flavoring, coloring, and sweetening agents asappropriate. Preparations for oral administration may be suitablyformulated for slow release, controlled release, or sustained release ofa prophylactic or therapeutic agent(s).

The method of the invention may comprise pulmonary administration, e.g.,by use of an inhaler or nebulizer, of a composition formulated with anaerosolizing agent. See, e.g., U.S. Pat. Nos. 6,019,968, 5,985,320, 5,985,309, 5,934,272, 5,874,064, 5,855,913, 5,290,540, and 4,880,078; andPCT Publication Nos. WO 92/19244, WO 97/32572, WO 97/44013, WO 98/31346,and WO 99/66903, each of which is incorporated herein by reference theirentireties. In a specific embodiment, a binding protein of theinvention, combination therapy, and/or composition of the invention isadministered using Alkermes AIR® pulmonary drug delivery technology(Alkermes, Inc., Cambridge, Mass.).

The method of the invention may comprise administration of a compositionformulated for parenteral administration by injection (e.g., by bolusinjection or continuous infusion). Formulations for injection may bepresented in unit dosage form (e.g., in ampoules or in multi-dosecontainers) with an added preservative. The compositions may take suchforms as suspensions, solutions or emulsions in oily or aqueousvehicles, and may contain formulatory agents such as suspending,stabilizing and/or dispersing agents. Alternatively, the activeingredient may be in powder form for constitution with a suitablevehicle (e.g., sterile pyrogen-free water) before use.

The methods of the invention may additionally comprise of administrationof compositions formulated as depot preparations. Such long actingformulations may be administered by implantation (e.g., subcutaneouslyor intramuscularly) or by intramuscular injection. Thus, for example,the compositions may be formulated with suitable polymeric orhydrophobic materials (e.g., as an emulsion in an acceptable oil) or ionexchange resins, or as sparingly soluble derivatives (e.g., as asparingly soluble salt).

The methods of the invention encompass administration of compositionsformulated as neutral or salt forms. Pharmaceutically acceptable saltsinclude those formed with anions such as those derived fromhydrochloric, phosphoric, acetic, oxalic, tartaric acids, etc., andthose formed with cations such as those derived from sodium, potassium,ammonium, calcium, ferric hydroxides, isopropylamine, triethylamine,2-ethylamino ethanol, histidine, procaine, etc.

Generally, the ingredients of compositions are supplied eitherseparately or mixed together in unit dosage form, for example, as a drylyophilized powder or water free concentrate in a hermetically sealedcontainer such as an ampoule or sachette indicating the quantity ofactive agent. Where the mode of administration is infusion, compositioncan be dispensed with an infusion bottle containing sterilepharmaceutical grade water or saline. Where the mode of administrationis by injection, an ampoule of sterile water for injection or saline canbe provided so that the ingredients may be mixed prior toadministration.

In particular, the invention also provides that one or more of theprophylactic or therapeutic agents, or pharmaceutical compositions ofthe invention is packaged in a hermetically sealed container such as anampoule or sachette indicating the quantity of the agent. In oneembodiment, one or more of the prophylactic or therapeutic agents, orpharmaceutical compositions of the invention is supplied as a drysterilized lyophilized powder or water free concentrate in ahermetically sealed container and can be reconstituted (e.g., with wateror saline) to the appropriate concentration for administration to asubject. Preferably, one or more of the prophylactic or therapeuticagents or pharmaceutical compositions of the invention is supplied as adry sterile lyophilized powder in a hermetically sealed container at aunit dosage of at least 5 mg, more preferably at least 10 mg, at least15 mg, at least 25 mg, at least 35 mg, at least 45 mg, at least 50 mg,at least 75 mg, or at least 100 mg. The lyophilized prophylactic ortherapeutic agents or pharmaceutical compositions of the inventionshould be stored at between 2° C. and 8° C. in its original containerand the prophylactic or therapeutic agents, or pharmaceuticalcompositions of the invention should be administered within 1 week,preferably within 5 days, within 72 hours, within 48 hours, within 24hours, within 12 hours, within 6 hours, within 5 hours, within 3 hours,or within 1 hour after being reconstituted. In an alternativeembodiment, one or more of the prophylactic or therapeutic agents orpharmaceutical compositions of the invention is supplied in liquid formin a hermetically sealed container indicating the quantity andconcentration of the agent. Preferably, the liquid form of theadministered composition is supplied in a hermetically sealed containerat least 0.25 mg/ml, more preferably at least 0.5 mg/ml, at least 1mg/ml, at least 2.5 mg/ml, at least 5 mg/ml, at least 8 mg/ml, at least10 mg/ml, at least 15 mg/kg, at least 25 mg/ml, at least 50 mg/ml, atleast 75 mg/ml or at least 100 mg/ml. The liquid form should be storedat between 2° C. and 8° C. in its original container.

The binding proteins of the invention can be incorporated into apharmaceutical composition suitable for parenteral administration.Preferably, the antibody or antibody-portions will be prepared as aninjectable solution containing 0.1-250 mg/ml binding protein. Theinjectable solution can be composed of either a liquid or lyophilizeddosage form in a flint or amber vial, ampule or pre-filled syringe. Thebuffer can be L-histidine (1-50 mM), optimally 5-10 mM, at pH 5.0 to 7.0(optimally pH 6.0). Other suitable buffers include but are not limitedto, sodium succinate, sodium citrate, sodium phosphate or potassiumphosphate. Sodium chloride can be used to modify the toxicity of thesolution at a concentration of 0-300 mM (optimally 150 mM for a liquiddosage form). Cryoprotectants can be included for a lyophilized dosageform, principally 0-10% sucrose (optimally 0.5-1.0%). Other suitablecryoprotectants include trehalose and lactose. Bulking agents can beincluded for a lyophilized dosage form, principally 1-10% mannitol(optimally 2-4%). Stabilizers can be used in both liquid and lyophilizeddosage forms, principally 1-50 mM L-Methionine (optimally 5-10 mM).Other suitable bulking agents include glycine, arginine, can be includedas 0-0.05% polysorbate-80 (optimally 0.005-0.01%). Additionalsurfactants include but are not limited to polysorbate 20 and BRIJsurfactants. The pharmaceutical composition comprising the bindingproteins of the invention prepared as an injectable solution forparenteral administration, can further comprise an agent useful as anadjuvant, such as those used to increase the absorption, or dispersionof a therapeutic protein (e.g., antibody). A particularly usefuladjuvant is hyaluronidase, such as HYLENEX® (recombinant humanhyaluronidase). Addition of hyaluronidase in the injectable solutionimproves human bioavailability following parenteral administration,particularly subcutaneous administration. It also allows for greaterinjection site volumes (i.e. greater than 1 ml) with less pain anddiscomfort, and minimum incidence of injection site reactions. (seeWO2004078140, and US2006104968 incorporated herein by reference).

The compositions of this invention may be in a variety of forms. Theseinclude, for example, liquid, semi-solid and solid dosage forms, such asliquid solutions (e.g., injectable and infusible solutions), dispersionsor suspensions, tablets, pills, powders, liposomes and suppositories.The preferred form depends on the intended mode of administration andtherapeutic application. Typical preferred compositions are in the formof injectable or infusible solutions, such as compositions similar tothose used for passive immunization of humans with other antibodies. Thepreferred mode of administration is parenteral (e.g., intravenous,subcutaneous, intraperitoneal, intramuscular). In a preferredembodiment, the antibody is administered by intravenous infusion orinjection. In another preferred embodiment, the antibody is administeredby intramuscular or subcutaneous injection.

Therapeutic compositions typically must be sterile and stable under theconditions of manufacture and storage. The composition can be formulatedas a solution, microemulsion, dispersion, liposome, or other orderedstructure suitable to high drug concentration. Sterile injectablesolutions can be prepared by incorporating the active compound (i.e.,antibody or antibody portion) in the required amount in an appropriatesolvent with one or a combination of ingredients enumerated above, asrequired, followed by filtered sterilization. Generally, dispersions areprepared by incorporating the active compound into a sterile vehiclethat contains a basic dispersion medium and the required otheringredients from those enumerated above. In the case of sterile,lyophilized powders for the preparation of sterile injectable solutions,the preferred methods of preparation are vacuum drying and spray-dryingthat yields a powder of the active ingredient plus any additionaldesired ingredient from a previously sterile-filtered solution thereof.The proper fluidity of a solution can be maintained, for example, by theuse of a coating such as lecithin, by the maintenance of the requiredparticle size in the case of dispersion and by the use of surfactants.Prolonged absorption of injectable compositions can be brought about byincluding, in the composition, an agent that delays absorption, forexample, monostearate salts and gelatin.

The binding proteins of the present invention can be administered by avariety of methods known in the art, although for many therapeuticapplications, the preferred route/mode of administration is subcutaneousinjection, intravenous injection or infusion. As will be appreciated bythe skilled artisan, the route and/or mode of administration will varydepending upon the desired results. In certain embodiments, the activecompound may be prepared with a carrier that will protect the compoundagainst rapid release, such as a controlled release formulation,including implants, transdermal patches, and microencapsulated deliverysystems. Biodegradable, biocompatible polymers can be used, such asethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen,polyorthoesters, and polylactic acid. Many methods for the preparationof such formulations are patented or generally known to those skilled inthe art. See, e.g., Sustained and Controlled Release Drug DeliverySystems, J. R. Robinson, ed., Marcel Dekker, Inc., New York, 1978.

In certain embodiments, a binding protein of the invention may be orallyadministered, for example, with an inert diluent or an assimilableedible carrier. The compound (and other ingredients, if desired) mayalso be enclosed in a hard or soft shell gelatin capsule, compressedinto tablets, or incorporated directly into the subject's diet. For oraltherapeutic administration, the compounds may be incorporated withexcipients and used in the form of ingestible tablets, buccal tablets,troches, capsules, elixirs, suspensions, syrups, wafers, and the like.To administer a compound of the invention by other than parenteraladministration, it may be necessary to coat the compound with, orco-administer the compound with, a material to prevent its inactivation.

Supplementary active compounds can also be incorporated into thecompositions. In certain embodiments, a binding protein of the inventionis coformulated with and/or coadministered with one or more additionaltherapeutic agents that are useful for treating disorders in which IL-12activity is detrimental. For example, a binding protein of the inventionmay be coformulated and/or coadministered with one or more additionalantibodies that bind other targets (e.g., antibodies that bind othercytokines or that bind cell surface molecules). Furthermore, one or moreantibodies of the invention may be used in combination with two or moreof the foregoing therapeutic agents. Such combination therapies mayadvantageously utilize lower dosages of the administered therapeuticagents, thus avoiding possible toxicities or complications associatedwith the various monotherapies.

In certain embodiments, a binding protein is linked to a half-lifeextending vehicle known in the art. Such vehicles include, but are notlimited to, the Fc domain, polyethylene glycol, and dextran. Suchvehicles are described, e.g., in U.S. application Ser. No. 09/428,082and published PCT Application No. WO 99/25044, which are herebyincorporated by reference for any purpose.

In a specific embodiment, nucleic acid sequences encoding a bindingprotein of the invention or another prophylactic or therapeutic agent ofthe invention are administered to treat, prevent, manage, or amelioratea disorder or one or more symptoms thereof by way of gene therapy. Genetherapy refers to therapy performed by the administration to a subjectof an expressed or expressible nucleic acid. In this embodiment of theinvention, the nucleic acids produce their encoded antibody orprophylactic or therapeutic agent of the invention that mediates aprophylactic or therapeutic effect.

Any of the methods for gene therapy available in the art can be usedaccording to the present invention. For general reviews of the methodsof gene therapy, see Goldspiel et al., 1993, Clinical Pharmacy12:488-505; Wu and Wu, 1991, Biotherapy 3:87-95; Tolstoshev, 1993, Ann.Rev. Pharmacol. Toxicol. 32:573-596; Mulligan, Science 260:926-932(1993); and Morgan and Anderson, 1993, Ann. Rev. Biochem. 62:191-217;May, 1993, TIBTECH 11(5):155-215. Methods commonly known in the art ofrecombinant DNA technology which can be used are described in Ausubel etal. (eds.), Current Protocols in Molecular Biology, John Wiley &Sons, NY(1993); and Kriegler, Gene Transfer and Expression, A Laboratory Manual,Stockton Press, NY (1990). Detailed description of various methods ofgene therapy are disclosed in US20050042664 A1 which is incorporatedherein by reference.

The binding proteins of the invention are useful in treating variousdiseases wherein the targets that are recognized by the binding proteinsare detrimental. Such diseases include, but are not limited to,rheumatoid arthritis, osteoarthritis, juvenile chronic arthritis, septicarthritis, Lyme arthritis, psoriatic arthritis, reactive arthritis,spondyloarthropathy, systemic lupus erythematosus, Crohn's disease,ulcerative colitis, inflammatory bowel disease, insulin dependentdiabetes mellitus, thyroiditis, asthma, allergic diseases, psoriasis,dermatitis scleroderma, graft versus host disease, organ transplantrejection, acute or chronic immune disease associated with organtransplantation, sarcoidosis, atherosclerosis, disseminatedintravascular coagulation, Kawasaki's disease, Grave's disease,nephrotic syndrome, chronic fatigue syndrome, Wegener's granulomatosis,Henoch-Schoenlein purpurea, microscopic vasculitis of the kidneys,chronic active hepatitis, uveitis, septic shock, toxic shock syndrome,sepsis syndrome, cachexia, infectious diseases, parasitic diseases,acquired immunodeficiency syndrome, acute transverse myelitis,Huntington's chorea, Parkinson's disease, Alzheimer's disease, stroke,primary biliary cirrhosis, hemolytic anemia, malignancies, heartfailure, myocardial infarction, Addison's disease, sporadic,polyglandular deficiency type I and polyglandular deficiency type II,Schmidt's syndrome, adult (acute) respiratory distress syndrome,alopecia, alopecia greata, seronegative arthopathy, arthropathy,Reiter's disease, psoriatic arthropathy, ulcerative colitic arthropathy,enteropathic synovitis, chlamydia, yersinia and salmonella associatedarthropathy, spondyloarthopathy, atheromatous disease/arteriosclerosis,atopic allergy, autoimmune bullous disease, pemphigus vulgaris,pemphigus foliaceus, pemphigoid, linear IgA disease, autoimmunehaemolytic anaemia, Coombs positive haemolytic anaemia, acquiredpernicious anaemia, juvenile pernicious anaemia, myalgicencephalitis/Royal Free Disease, chronic mucocutaneous candidiasis,giant cell arteritis, primary sclerosing hepatitis, cryptogenicautoimmune hepatitis, Acquired Immunodeficiency Disease Syndrome,Acquired Immunodeficiency Related Diseases, Hepatitis B, Hepatitis C,common varied immunodeficiency (common variable hypogammaglobulinaemia),dilated cardiomyopathy, female infertility, ovarian failure, prematureovarian failure, fibrotic lung disease, cryptogenic fibrosingalveolitis, post-inflammatory interstitial lung disease, interstitialpneumonitis, connective tissue disease associated interstitial lungdisease, mixed connective tissue disease associated lung disease,systemic sclerosis associated interstitial lung disease, rheumatoidarthritis associated interstitial lung disease, systemic lupuserythematosus associated lung disease, dermatomyositis/polymyositisassociated lung disease, Sjögren's disease associated lung disease,ankylosing spondylitis associated lung disease, vasculitic diffuse lungdisease, haemosiderosis associated lung disease, drug-inducedinterstitial lung disease, fibrosis, radiation fibrosis, bronchiolitisobliterans, chronic eosinophilic pneumonia, lymphocytic infiltrativelung disease, postinfectious interstitial lung disease, gouty arthritis,autoimmune hepatitis, type-1 autoimmune hepatitis (classical autoimmuneor lupoid hepatitis), type-2 autoimmune hepatitis (anti-LKM antibodyhepatitis), autoimmune mediated hypoglycaemia, type B insulin resistancewith acanthosis nigricans, hypoparathyroidism, acute immune diseaseassociated with organ transplantation, chronic immune disease associatedwith organ transplantation, osteoarthrosis, primary sclerosingcholangitis, psoriasis type 1, psoriasis type 2, idiopathic leucopaenia,autoimmune neutropaenia, renal disease NOS, glomerulonephritides,microscopic vasulitis of the kidneys, lyme disease, discoid lupuserythematosus, male infertility idiopathic or NOS, sperm autoimmunity,multiple sclerosis (all subtypes), sympathetic ophthalmia, pulmonaryhypertension secondary to connective tissue disease, Goodpasture'ssyndrome, pulmonary manifestation of polyarteritis nodosa, acuterheumatic fever, rheumatoid spondylitis, Still's disease, systemicsclerosis, Sjörgren's syndrome, Takayasu's disease/arteritis, autoimmunethrombocytopaenia, idiopathic thrombocytopaenia, autoimmune thyroiddisease, hyperthyroidism, goitrous autoimmune hypothyroidism(Hashimoto's disease), atrophic autoimmune hypothyroidism, primarymyxoedema, phacogenic uveitis, primary vasculitis, vitiligo acute liverdisease, chronic liver diseases, alcoholic cirrhosis, alcohol-inducedliver injury, choleosatatis, idiosyncratic liver disease, Drug-inducedhepatitis, Non-alcoholic Steatohepatitis, allergy and asthma, group Bstreptococci (GBS) infection, mental disorders (e.g., depression andschizophrenia), Th2 Type and Th1 Type mediated diseases, acute andchronic pain (different forms of pain), and cancers such as lung,breast, stomach, bladder, colon, pancreas, ovarian, prostate and rectalcancer and hematopoietic malignancies (leukemia and lymphoma),Abetalipoprotemia, Acrocyanosis, acute and chronic parasitic orinfectious processes, acute leukemia, acute lymphoblastic leukemia(ALL), acute myeloid leukemia (AML), acute or chronic bacterialinfection, acute pancreatitis, acute renal failure, adenocarcinomas,aerial ectopic beats, AIDS dementia complex, alcohol-induced hepatitis,allergic conjunctivitis, allergic contact dermatitis, allergic rhinitis,allograft rejection, alpha-1-antitrypsin deficiency, amyotrophic lateralsclerosis, anemia, angina pectoris, anterior horn cell degeneration,anti cd3 therapy, antiphospholipid syndrome, anti-receptorhypersensitivity reactions, aordic and peripheral aneuryisms, aorticdissection, arterial hypertension, arteriosclerosis, arteriovenousfistula, ataxia, atrial fibrillation (sustained or paroxysmal), atrialflutter, atrioventricular block, B cell lymphoma, bone graft rejection,bone marrow transplant (BMT) rejection, bundle branch block, Burkitt'slymphoma, Burns, cardiac arrhythmias, cardiac stun syndrome, cardiactumors, cardiomyopathy, cardiopulmonary bypass inflammation response,cartilage transplant rejection, cerebellar cortical degenerations,cerebellar disorders, chaotic or multifocal atrial tachycardia,chemotherapy associated disorders, chromic myelocytic leukemia (CML),chronic alcoholism, chronic inflammatory pathologies, chroniclymphocytic leukemia (CLL), chronic obstructive pulmonary disease(COPD), chronic salicylate intoxication, colorectal carcinoma,congestive heart failure, conjunctivitis, contact dermatitis, corpulmonale, coronary artery disease, Creutzfeldt-Jakob disease, culturenegative sepsis, cystic fibrosis, cytokine therapy associated disorders,Dementia pugilistica, demyelinating diseases, dengue hemorrhagic fever,dermatitis, dermatologic conditions, diabetes, diabetes mellitus,diabetic ateriosclerotic disease, Diffuse Lewy body disease, dilatedcongestive cardiomyopathy, disorders of the basal ganglia, Down'sSyndrome in middle age, drug-induced movement disorders induced by drugswhich block CNS dopamine receptors, drug sensitivity, eczema,encephalomyelitis, endocarditis, endocrinopathy, epiglottitis,epstein-barr virus infection, erythromelalgia, extrapyramidal andcerebellar disorders, familial hematophagocytic lymphohistiocytosis,fetal thymus implant rejection, Friedreich's ataxia, functionalperipheral arterial disorders, fungal sepsis, gas gangrene, gastriculcer, glomerular nephritis, graft rejection of any organ or tissue,gram negative sepsis, gram positive sepsis, granulomas due tointracellular organisms, hairy cell leukemia, Hallerrorden-Spatzdisease, hashimoto's thyroiditis, hay fever, heart transplant rejection,hemachromatosis, hemodialysis, hemolytic uremic syndrome/thrombolyticthrombocytopenic purpura, hemorrhage, hepatitis (A), His bundlearrythmias, HIV infection/HIV neuropathy, Hodgkin's disease,hyperkinetic movement disorders, hypersensitity reactions,hypersensitivity pneumonitis, hypertension, hypokinetic movementdisorders, hypothalamic-pituitary-adrenal axis evaluation, idiopathicAddison's disease, idiopathic pulmonary fibrosis, antibody mediatedcytotoxicity, Asthenia, infantile spinal muscular atrophy, inflammationof the aorta, influenza a, ionizing radiation exposure,iridocyclitis/uveitis/optic neuritis, ischemia-reperfusion injury,ischemic stroke, juvenile rheumatoid arthritis, juvenile spinal muscularatrophy, Kaposi's sarcoma, kidney transplant rejection, legionella,leishmaniasis, leprosy, lesions of the corticospinal system, lipedema,liver transplant rejection, lymphederma, malaria, malignamt Lymphoma,malignant histiocytosis, malignant melanoma, meningitis,meningococcemia, metabolic/idiopathic, migraine headache, mitochondrialmulti.system disorder, mixed connective tissue disease, monoclonalgammopathy, multiple myeloma, multiple systems degenerations (MencelDejerine-Thomas Shi-Drager and Machado-Joseph), myasthenia gravis,mycobacterium avium intracellulare, mycobacterium tuberculosis,myelodyplastic syndrome, myocardial infarction, myocardial ischemicdisorders, nasopharyngeal carcinoma, neonatal chronic lung disease,nephritis, nephrosis, neurodegenerative diseases, neurogenic I muscularatrophies, neutropenic fever, non-hodgkins lymphoma, occlusion of theabdominal aorta and its branches, occulsive arterial disorders, okt3therapy, orchitis/epidydimitis, orchitis/vasectomy reversal procedures,organomegaly, osteoporosis, pancreas transplant rejection, pancreaticcarcinoma, paraneoplastic syndrome/hypercalcemia of malignancy,parathyroid transplant rejection, pelvic inflammatory disease, perennialrhinitis, pericardial disease, peripheral atherlosclerotic disease,peripheral vascular disorders, peritonitis, pernicious anemia,pneumocystis carinii pneumonia, pneumonia, POEMS syndrome(polyneuropathy, organomegaly, endocrinopathy, monoclonal gammopathy,and skin changes syndrome), post perfusion syndrome, post pump syndrome,post-MI cardiotomy syndrome, preeclampsia, Progressive supranucleoPalsy, primary pulmonary hypertension, radiation therapy, Raynaud'sphenomenon and disease, Raynoud's disease, Refsum's disease, regularnarrow QRS tachycardia, renovascular hypertension, reperfusion injury,restrictive cardiomyopathy, sarcomas, scleroderma, senile chorea, SenileDementia of Lewy body type, seronegative arthropathies, shock, sicklecell anemia, skin allograft rejection, skin changes syndrome, smallbowel transplant rejection, solid tumors, specific arrythmias, spinalataxia, spinocerebellar degenerations, streptococcal myositis,structural lesions of the cerebellum, Subacute sclerosingpanencephalitis, Syncope, syphilis of the cardiovascular system,systemic anaphalaxis, systemic inflammatory response syndrome, systemiconset juvenile rheumatoid arthritis, T-cell or FAB ALL, Telangiectasia,thromboangitis obliterans, thrombocytopenia, toxicity, transplants,trauma/hemorrhage, type III hypersensitivity reactions, type IVhypersensitivity, unstable angina, uremia, urosepsis, urticaria,valvular heart diseases, varicose veins, vasculitis, venous diseases,venous thrombosis, ventricular fibrillation, viral and fungalinfections, vital encephalitis/aseptic meningitis, vital-associatedhemaphagocytic syndrome, Wernicke-Korsakoff syndrome, Wilson's disease,xenograft rejection of any organ or tissue. (see Peritt et al. PCTpublication No. WO2002097048A2, Leonard et al., PCT publication No.WO9524918 A1, and Salfeld et al., PCT publication No. WO00/56772A 1).

The binding proteins of the invention can be used to treat humanssuffering from autoimmune diseases, in particular those associated withinflammation, including, rheumatoid arthritis, spondylitis, allergy,autoimmune diabetes, autoimmune uveitis.

Preferably, the binding proteins of the invention or antigen-bindingportions thereof, are used to treat rheumatoid arthritis, Crohn'sdisease, multiple sclerosis, insulin dependent diabetes mellitus andpsoriasis.

A binding protein of the invention also can be administered with one ormore additional therapeutic agents useful in the treatment of variousdiseases.

A binding protein of the invention can be used alone or in combinationto treat such diseases. It should be understood that the bindingproteins can be used alone or in combination with an additional agent,e.g., a therapeutic agent, said additional agent being selected by theskilled artisan for its intended purpose. For example, the additionalagent can be a therapeutic agent art-recognized as being useful to treatthe disease or condition being treated by the antibody of the presentinvention. The additional agent also can be an agent that imparts abeneficial attribute to the therapeutic composition e.g., an agent whicheffects the viscosity of the composition.

It should further be understood that the combinations which are to beincluded within this invention are those combinations useful for theirintended purpose. The agents set forth below are illustrative forpurposes and not intended to be limited. The combinations, which arepart of this invention, can be the antibodies of the present inventionand at least one additional agent selected from the lists below. Thecombination can also include more than one additional agent, e.g., twoor three additional agents if the combination is such that the formedcomposition can perform its intended function.

Preferred combinations to treat autoimmune and inflammatory diseases arenon-steroidal anti-inflammatory drug(s) also referred to as NSAIDS whichinclude drugs like ibuprofen. Other preferred combinations arecorticosteroids including prednisolone; the well known side-effects ofsteroid use can be reduced or even eliminated by tapering the steroiddose required when treating patients in combination with the DVD Igs ofthis invention. Non-limiting examples of therapeutic agents forrheumatoid arthritis with which an antibody, or antibody portion, of theinvention can be combined include the following: cytokine suppressiveanti-inflammatory drug(s) (CSAIDs); antibodies to or antagonists ofother human cytokines or growth factors, for example, TNF, LT, IL-1,IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-15, IL-16, IL-18, IL-21,IL-23, interferons, EMAP-II, GM-CSF, FGF, and PDGF. Binding proteins ofthe invention, or antigen binding portions thereof, can be combined withantibodies to cell surface molecules such as CD2, CD3, CD4, CD8, CD25,CD28, CD30, CD40, CD45, CD69, CD80 (B7.1), CD86 (B7.2), CD90, CTLA ortheir ligands including CD154 (gp39 or CD40L).

Preferred combinations of therapeutic agents may interfere at differentpoints in the autoimmune and subsequent inflammatory cascade; preferredexamples include TNF antagonists like chimeric, humanized or human TNFantibodies, D2E7, (PCT Publication No. WO 97/29131), CA2 (REMICADE®infliximab), CDP 571, and soluble p55 or p75 TNF receptors, derivatives,thereof, (p75TNFR1gG (ENBREL® etanercept) or p55TNFR1gG (lenercept), andalso TNFα converting enzyme (TACE) inhibitors; similarly IL-1 inhibitors(Interleukin-1-converting enzyme inhibitors, IL-1RA etc.) may beeffective for the same reason. Other preferred combinations includeInterleukin 11. Yet another preferred combination include key players ofthe autoimmune response which may act parallel to, dependent on or inconcert with IL-12 function; especially preferred are IL-18 antagonistsincluding IL-18 antibodies or soluble IL-18 receptors, or IL-18 bindingproteins. It has been shown that IL-12 and IL-18 have overlapping butdistinct functions and a combination of antagonists to both may be mosteffective. Yet another preferred combination are non-depleting anti-CD4inhibitors. Yet other preferred combinations include antagonists of theco-stimulatory pathway CD80 (B7.1) or CD86 (B7.2) including antibodies,soluble receptors or antagonistic ligands.

The binding proteins of the invention may also be combined with agents,such as methotrexate, 6-MP, azathioprine sulphasalazine, mesalazine,olsalazine chloroquinine/hydroxychloroquine, pencillamine,aurothiomalate (intramuscular and oral), azathioprine, cochicine,corticosteroids (oral, inhaled and local injection), beta-2adrenoreceptor agonists (salbutamol, terbutaline, salmeteral), xanthines(theophylline, aminophylline), cromoglycate, nedocromil, ketotifen,ipratropium and oxitropium, cyclosporin, FK506, rapamycin, mycophenolatemofetil, leflunomide, NSAIDs, for example, ibuprofen, corticosteroidssuch as prednisolone, phosphodiesterase inhibitors, adensosine agonists,antithrombotic agents, complement inhibitors, adrenergic agents, agentswhich interfere with signalling by proinflammatory cytokines such asTNF□ or IL-1 (e.g. IRAK, NIK, IKK, p38 or MAP kinase inhibitors), IL-1βconverting enzyme inhibitors, TNFαxconverting enzyme (TACE) inhibitors,T-cell signalling inhibitors such as kinase inhibitors,metalloproteinase inhibitors, sulfasalazine, azathioprine,6-mercaptopurines, angiotensin converting enzyme inhibitors, solublecytokine receptors and derivatives thereof (e.g. soluble p55 or p75 TNFreceptors and the derivatives p75TNFRIgG (ENBREL® etanercept andp55TNFRIgG (lenercept)), sIL-1RI, sIL-1RII, sIL-6R), antiinflammatorycytokines (e.g. IL-4, IL-10, IL-11, IL-13 and TGFβ), celecoxib, folicacid, hydroxychloroquine sulfate, rofecoxib, etanercept, infliximab,naproxen, valdecoxib, sulfasalazine, methylprednisolone, meloxicam,methylprednisolone acetate, gold sodium thiomalate, aspirin,triamcinolone acetonide, propoxyphene napsylate/apap, folate,nabumetone, diclofenac, piroxicam, etodolac, diclofenac sodium,oxaprozin, oxycodone hcl, hydrocodone bitartrate/apap, diclofenacsodium/misoprostol, fentanyl, anakinra, human recombinant, tramadol hcl,salsalate, sulindac, cyanocobalamin/fa/pyridoxine, acetaminophen,alendronate sodium, prednisolone, morphine sulfate, lidocainehydrochloride, indomethacin, glucosamine sulf/chondroitin, amitriptylinehcl, sulfadiazine, oxycodone hcl/acetaminophen, olopatadine hcl,misoprostol, naproxen sodium, omeprazole, cyclophosphamide, rituximab,IL-1 TRAP, MRA, CTLA4-IG, IL-18 BP, anti-IL-18, Anti-IL15, BIRB-796,SCIO-469, VX-702, AMG-548, VX-740, Roflumilast, IC-485, CDC-801, andMesopram. Preferred combinations include methotrexate or leflunomide andin moderate or severe rheumatoid arthritis cases, cyclosporine.

Nonlimiting additional agents which can also be used in combination witha binding protein to treat rheumatoid arthritis include, but are notlimited to, the following: non-steroidal anti-inflammatory drug(s)(NSAIDs); cytokine suppressive anti-inflammatory drug(s) (CSAIDs);CDP-571/BAY-10-3356 (humanized anti-TNFα antibody; Celltech/Bayer);cA2/infliximab (chimeric anti-TNFαc antibody; Centocor); 75kdTNFR-IgG/etanercept (75 kD TNF receptor-IgG fusion protein; Immunex;see e.g., Arthritis & Rheumatism (1994) Vol. 37, S295; J. Invest. Med.L(1996) Vol. 44, 235A); 55 kdTNF-IgG (55 kD TNF receptor-IgG fusionprotein; Hoffmann-LaRoche); IDEC-CE9.1/SB 210396 (non-depletingprimatized anti-CD4 antibody; IDEC/SmithKline; see e.g., Arthritis &Rheumatism (1995) Vol. 38, S185); DAB 486-IL-2 and/or DAB 389-IL-2 (IL-2fusion proteins; Seragen; see e.g., Arthritis & Rheumatism (1993) Vol.36, 1223); Anti-Tac (humanized anti-IL-2Rα; Protein Design Labs/Roche);IL-4 (anti-inflammatory cytokine; DNAX/Schering); IL-10 (SCH 52000;recombinant IL-10, anti-inflammatory cytokine; DNAX/Schering); IL-4;IL-10 and/or IL-4 agonists (e.g., agonist antibodies); IL-1RA (IL-1receptor antagonist; Synergen/Amgen); anakinra (KINERET®/Amgen);TNF-bp/s-TNF (soluble TNF binding protein; see e.g., Arthritis &Rheumatism (1996) Vol. 39, No. 9 (supplement), S284; Amer. J.Physiol.—Heart and Circulatory Physiology (1995) Vol. 268, pp. 37-42);R973401 (phosphodiesterase Type IV inhibitor; see e.g., Arthritis &Rheumatism (1996) Vol. 39, No. 9 (supplement), S282); MK-966 (COX-2Inhibitor; see e.g., Arthritis & Rheumatism (1996) Vol. 39, No. 9(supplement), S81); Iloprost (see e.g., Arthritis & Rheumatism (1996)Vol. 39, No. 9 (supplement), S82); methotrexate; thalidomide (see e.g.,Arthritis & Rheumatism (1996) Vol. 39, No. 9 (supplement), S282) andthalidomide-related drugs (e.g., Celgen); leflunomide (anti-inflammatoryand cytokine inhibitor; see e.g., Arthritis & Rheumatism (1996) Vol. 39,No. 9 (supplement), S131; Inflammation Research (1996) Vol. 45, pp.103-107); tranexamic acid (inhibitor of plasminogen activation; seee.g., Arthritis & Rheumatism (1996) Vol. 39, No. 9 (supplement), S284);T-614 (cytokine inhibitor; see e.g., Arthritis & Rheumatism (1996) Vol.39, No. 9 (supplement), S282); prostaglandin E1 (see e.g., Arthritis &Rheumatism (1996) Vol. 39, No. 9 (supplement), S282); Tenidap(non-steroidal anti-inflammatory drug; see e.g., Arthritis & Rheumatism(1996) Vol. 39, No. 9 (supplement), S280); Naproxen (non-steroidalanti-inflammatory drug; see e.g., Neuro Report (1996) Vol. 7, pp.1209-1213); Meloxicam (non-steroidal anti-inflammatory drug); Ibuprofen(non-steroidal anti-inflammatory drug); Piroxicam (non-steroidalanti-inflammatory drug); Diclofenac (non-steroidal anti-inflammatorydrug); Indomethacin (non-steroidal anti-inflammatory drug);Sulfasalazine (see e.g., Arthritis & Rheumatism (1996) Vol. 39, No. 9(supplement), S281); Azathioprine (see e.g., Arthritis & Rheumatism(1996) Vol. 39, No. 9 (supplement), S281); ICE inhibitor (inhibitor ofthe enzyme interleukin-1β converting enzyme); zap-70 and/or lckinhibitor (inhibitor of the tyrosine kinase zap-70 or lck); VEGFinhibitor and/or VEGF-R inhibitor (inhibitors of vascular endothelialcell growth factor or vascular endothelial cell growth factor receptor;inhibitors of angiogenesis); corticosteroid anti-inflammatory drugs(e.g., SB203580); TNF-convertase inhibitors; anti-IL-12 antibodies;anti-IL-18 antibodies; interleukin-11 (see e.g., Arthritis & Rheumatism(1996) Vol. 39, No. 9 (supplement), S296); interleukin-13 (see e.g.,Arthritis & Rheumatism (1996) Vol. 39, No. 9 (supplement), S308);interleukin-17 inhibitors (see e.g., Arthritis & Rheumatism (1996) Vol.39, No. 9 (supplement), S120); gold; penicillamine; chloroquine;chlorambucil; hydroxychloroquine; cyclosporine; cyclophosphamide; totallymphoid irradiation; anti-thymocyte globulin; anti-CD4 antibodies;CD5-toxins; orally-administered peptides and collagen; lobenzaritdisodium; Cytokine Regulating Agents (CRAs) HP228 and HP466 (HoughtenPharmaceuticals, Inc.); ICAM-1 antisense phosphorothioateoligo-deoxynucleotides (ISIS 2302; Isis Pharmaceuticals, Inc.); solublecomplement receptor 1 (TP10; T Cell Sciences, Inc.); prednisone;orgotein; glycosaminoglycan polysulphate; minocycline; anti-IL2Rantibodies; marine and botanical lipids (fish and plant seed fattyacids; see e.g., DeLuca et al. (1995) Rheum. Dis. Clin. North Am.21:759-777); auranofin; phenylbutazone; meclofenamic acid; flufenamicacid; intravenous immune globulin; zileuton; azaribine; mycophenolicacid (RS-61443); tacrolimus (FK-506); sirolimus (rapamycin); amiprilose(therafectin); cladribine (2-chlorodeoxyadenosine); methotrexate;antivirals; and immune modulating agents.

In one embodiment, the binding protein or antigen-binding portionthereof, is administered in combination with one of the following agentsfor the treatment of rheumatoid arthritis: small molecule inhibitor ofKDR (ABT-123), small molecule inhibitor of Tie-2; methotrexate;prednisone; celecoxib; folic acid; hydroxychloroquine sulfate;rofecoxib; etanercept; infliximab; leflunomide; naproxen; valdecoxib;sulfasalazine; methylprednisolone; ibuprofen; meloxicam;methylprednisolone acetate; gold sodium thiomalate; aspirin;azathioprine; triamcinolone acetonide; propxyphene napsylate/apap;folate; nabumetone; diclofenac; piroxicam; etodolac; diclofenac sodium;oxaprozin; oxycodone hcl; hydrocodone bitartrate/apap; diclofenacsodium/misoprostol; fentanyl; anakinra, human recombinant; tramadol hcl;salsalate; sulindac; cyanocobalamin/fa/pyridoxine; acetaminophen;alendronate sodium; prednisolone; morphine sulfate; lidocainehydrochloride; indomethacin; glucosamine sulfate/chondroitin;cyclosporine; amitriptyline hcl; sulfadiazine; oxycodonehcl/acetaminophen; olopatadine hcl; misoprostol; naproxen sodium;omeprazole; mycophenolate mofetil; cyclophosphamide; rituximab; IL-1TRAP; MRA; CTLA4-IG; IL-18 BP; ABT-874; ABT-325 (anti-IL 18); anti-IL15; BIRB-796; SCIO-469; VX-702; AMG-548; VX-740; roflumilast; IC-485;CDC-801; and mesopram.

Non-limiting examples of therapeutic agents for inflammatory boweldisease with which a binding protein of the invention can be combinedinclude the following: budenoside; epidermal growth factor;corticosteroids; cyclosporin, sulfasalazine; aminosalicylates;6-mercaptopurine; azathioprine; metronidazole; lipoxygenase inhibitors;mesalamine; olsalazine; balsalazide; antioxidants; thromboxaneinhibitors; IL-1 receptor antagonists; anti-IL-1β monoclonal antibodies;anti-IL-6 monoclonal antibodies; growth factors; elastase inhibitors;pyridinyl-imidazole compounds; antibodies to or antagonists of otherhuman cytokines or growth factors, for example, TNF, LT, IL-1, IL-2,IL-6, IL-7, IL-8, IL-15, IL-16, IL-17, IL-18, EMAP-II, GM-CSF, FGF, andPDGF. Antibodies of the invention, or antigen binding portions thereof,can be combined with antibodies to cell surface molecules such as CD2,CD3, CD4, CD8, CD25, CD28, CD30, CD40, CD45, CD69, CD90 or theirligands. The antibodies of the invention, or antigen binding portionsthereof, may also be combined with agents, such as methotrexate,cyclosporin, FK506, rapamycin, mycophenolate mofetil, leflunomide,NSAIDs, for example, ibuprofen, corticosteroids such as prednisolone,phosphodiesterase inhibitors, adenosine agonists, antithrombotic agents,complement inhibitors, adrenergic agents, agents which interfere withsignalling by proinflammatory cytokines such as TNFα or IL-1 (e.g. IRAK,NIK, IKK, p38 or MAP kinase inhibitors), IL-1β converting enzymeinhibitors, TNFα converting enzyme inhibitors, T-cell signallinginhibitors such as kinase inhibitors, metalloproteinase inhibitors,sulfasalazine, azathioprine, 6-mercaptopurines, angiotensin convertingenzyme inhibitors, soluble cytokine receptors and derivatives thereof(e.g. soluble p55 or p75 TNF receptors, sIL-1R1, sIL-1RII, sIL-6R) andantiinflammatory cytokines (e.g. IL-4, IL-10, IL-11, IL-13 and TGFβ).

Preferred examples of therapeutic agents for Crohn's disease in which abinding protein can be combined include the following: TNF antagonists,for example, anti-TNF antibodies, D2E7 (PCT Publication No. WO 97/29131;HUMIRA® adalimumab), CA2 (REMICADE® infliximab), CDP 571, TNFR-Igconstructs, (p75TNFRIgG (ENBREL® etanercept) and p55TNFRIgG (lenercept))inhibitors and PDE4 inhibitors. Antibodies of the invention, or antigenbinding portions thereof, can be combined with corticosteroids, forexample, budenoside and dexamethasone. Binding proteins of the inventionor antigen binding portions thereof, may also be combined with agentssuch as sulfasalazine, 5-aminosalicylic acid and olsalazine, and agentswhich interfere with synthesis or action of proinflammatory cytokinessuch as IL-1, for example, IL-1β converting enzyme inhibitors andIL-1ra. Antibodies of the invention or antigen binding portion thereofmay also be used with T cell signaling inhibitors, for example, tyrosinekinase inhibitors 6-mercaptopurines. Binding proteins of the invention,or antigen binding portions thereof, can be combined with IL-11. Bindingproteins of the invention, or antigen binding portions thereof, can becombined with mesalamine, prednisone, azathioprine, mercaptopurine,infliximab, methylprednisolone sodium succinate, diphenoxylate/atropsulfate, loperamide hydrochloride, methotrexate, omeprazole, folate,ciprofloxacin/dextrose-water, hydrocodone bitartrate/apap, tetracyclinehydrochloride, fluocinonide, metronidazole, thimerosal/boric acid,cholestyramine/sucrose, ciprofloxacin hydrochloride, hyoscyaminesulfate, meperidine hydrochloride, midazolam hydrochloride, oxycodonehcl/acetaminophen, promethazine hydrochloride, sodium phosphate,sulfamethoxazole/trimethoprim, celecoxib, polycarbophil, propoxyphenenapsylate, hydrocortisone, multivitamins, balsalazide disodium, codeinephosphate/apap, colesevelam hcl, cyanocobalamin, folic acid,levofloxacin, methylprednisolone, natalizumab and interferon-gamma.

Non-limiting examples of therapeutic agents for multiple sclerosis withwhich binding proteins of the invention can be combined include thefollowing: corticosteroids; prednisolone; methylprednisolone;azathioprine; cyclophosphamide; cyclosporine; methotrexate;4-aminopyridine; tizanidine; interferon-β1a (AVONEX®; Biogen);interferon-β1b (BETASERON®; Chiron/Berlex); interferon α-n3) (InterferonSciences/Fujimoto), interferon-α (Alfa Wassermann/J&J), interferonβ1A-IF (Serono/Inhale Therapeutics), Peginterferon α 2b(Enzon/Schering-Plough), Copolymer 1 (Cop-1; COPAXONE®; TevaPharmaceutical Industries, Inc.); hyperbaric oxygen; intravenousimmunoglobulin; clabribine; antibodies to or antagonists of other humancytokines or growth factors and their receptors, for example, TNF, LT,IL-1, IL-2, IL-6, IL-7, IL-8, IL-23, IL-15, IL-16, IL-18, EMAP-II,GM-CSF, FGF, and PDGF. Binding proteins of the invention can be combinedwith antibodies to cell surface molecules such as CD2, CD3, CD4, CD8,CD19, CD20, CD25, CD28, CD30, CD40, CD45, CD69, CD80, CD86, CD90 ortheir ligands. Binding proteins of the invention, may also be combinedwith agents, such as methotrexate, cyclosporine, FK506, rapamycin,mycophenolate mofetil, leflunomide, NSAIDs, for example, ibuprofen,corticosteroids such as prednisolone, phosphodiesterase inhibitors,adensosine agonists, antithrombotic agents, complement inhibitors,adrenergic agents, agents which interfere with signalling byproinflammatory cytokines such as TNFα or IL-1 (e.g. IRAK, NIK, IKK, p38or MAP kinase inhibitors), IL-1β converting enzyme inhibitors, TACEinhibitors, T-cell signaling inhibitors such as kinase inhibitors,metalloproteinase inhibitors, sulfasalazine, azathioprine,6-mercaptopurines, angiotensin converting enzyme inhibitors, solublecytokine receptors and derivatives thereof (e.g. soluble p55 or p75 TNFreceptors, sIL-1RI, sIL-1RII, sIL-6R) and antiinflammatory cytokines(e.g. IL-4, IL-10, IL-13 and TGFβ).

Preferred examples of therapeutic agents for multiple sclerosis in whichbinding proteins of the invention can be combined tinclude interferon-β,for example, IFNβ1a and IFNβ1b; copaxone, corticosteroids, caspaseinhibitors, for example inhibitors of caspase-1, IL-1 inhibitors, TNFinhibitors, and antibodies to CD40 ligand and CD80.

The binding proteins of the invention, may also be combined with agents,such as alemtuzumab, dronabinol, Unimed, daclizumab, mitoxantrone,xaliproden hydrochloride, fampridine, glatiramer acetate, natalizumab,sinnabidol, a-immunokine NNSO3, ABR-215062, AnergiX.MS, chemokinereceptor antagonists, BBR-2778, calagualine, CPI-1189, LEM (liposomeencapsulated mitoxantrone), THC.CBD (cannabinoid agonist) MBP-8298,mesopram (PDE4 inhibitor), MNA-715, anti-IL-6 receptor antibody,neurovax, pirfenidone allotrap 1258 (RDP-1258), sTNF-R1, talampanel,teriflunomide, TGF-beta2, tiplimotide, VLA-4 antagonists (for example,TR-14035, VLA4 Ultrahaler, ANTEGREN® natalizumab—ELAN/Biogen),interferon gamma antagonists, IL-4 agonists.

Non-limiting examples of therapeutic agents for Angina with whichbinding proteins of the invention can be combined include the following:aspirin, nitroglycerin, isosorbide mononitrate, metoprolol succinate,atenolol, metoprolol tartrate, amlodipine besylate, diltiazemhydrochloride, isosorbide dinitrate, clopidogrel bisulfate, nifedipine,atorvastatin calcium, potassium chloride, furosemide, simvastatin,verapamil hcl, digoxin, propranolol hydrochloride, carvedilol,lisinopril, spironolactone, hydrochlorothiazide, enalapril maleate,nadolol, ramipril, enoxaparin sodium, heparin sodium, valsartan, sotalolhydrochloride, fenofibrate, ezetimibe, bumetanide, losartan potassium,lisinopril/hydrochlorothiazide, felodipine, captopril, bisoprololfumarate.

Non-limiting examples of therapeutic agents for Ankylosing Spondylitiswith which binding proteins of the invention can be combined include thefollowing: ibuprofen, diclofenac and misoprostol, naproxen, meloxicam,indomethacin, diclofenac, celecoxib, rofecoxib, Sulfasalazine,Methotrexate, azathioprine, minocyclin, prednisone, etanercept,infliximab.

Non-limiting examples of therapeutic agents for Asthma with whichbinding proteins of the invention can be combined include the following:albuterol, salmeterol/fluticasone, montelukast sodium, fluticasonepropionate, budesonide, prednisone, salmeterol xinafoate, levalbuterolhcl, albuterol sulfate/ipratropium, prednisolone sodium phosphate,triamcinolone acetonide, beclomethasone dipropionate, ipratropiumbromide, azithromycin, pirbuterol acetate, prednisolone, theophyllineanhydrous, methylprednisolone sodium succinate, clarithromycin,zafirlukast, formoterol fumarate, influenza virus vaccine,methylprednisolone, amoxicillin trihydrate, flunisolide, allergyinjection, cromolyn sodium, fexofenadine hydrochloride,flunisolide/menthol, amoxicillin/clavulanate, levofloxacin, inhalerassist device, guaifenesin, dexamethasone sodium phosphate, moxifloxacinhcl, doxycycline hyclate, guaifenesin/d-methorphan,p-ephedrine/cod/chlorphenir, gatifloxacin, cetirizine hydrochloride,mometasone furoate, salmeterol xinafoate, benzonatate, cephalexin,pe/hydrocodone/chlorphenir, cetirizine hcl/pseudoephed,phenylephrine/cod/promethazine, codeine/promethazine, cefprozil,dexamethasone, guaifenesin/pseudoephedrine, chlorpheniramine/hydrocodone, nedocromil sodium, terbutaline sulfate,epinephrine, methylprednisolone, metaproterenol sulfate.

Non-limiting examples of therapeutic agents for COPD with which bindingproteins of the invention can be combined include the following:albuterol sulfate/ipratropium, ipratropium bromide,salmeterol/fluticasone, albuterol, salmeterol xinafoate, fluticasonepropionate, prednisone, theophylline anhydrous, methylprednisolonesodium succinate, montelukast sodium, budesonide, formoterol fumarate,triamcinolone acetonide, levofloxacin, guaifenesin, azithromycin,beclomethasone dipropionate, levalbuterol hcl, flunisolide, ceftriaxonesodium, amoxicillin trihydrate, gatifloxacin, zafirlukast,amoxicillin/clavulanate, flunisolide/menthol,chlorpheniramine/hydrocodone, metaproterenol sulfate,methylprednisolone, mometasone furoate, p-ephedrine/cod/chlorphenir,pirbuterol acetate, p-ephedrine/loratadine, terbutaline sulfate,tiotropium bromide, (R,R)-formoterol, TgAAT, Cilomilast, Roflumilast.

Non-limiting examples of therapeutic agents for HCV with which bindingproteins of the invention can be combined include the following:Interferon-alpha-2a, Interferon-alpha-2b, Interferon-alpha con1,Interferon-alpha-n1, Pegylated interferon-alpha-2a, Pegylatedinterferon-alpha-2b, ribavirin, Peginterferon alfa-2b+ribavirin,Ursodeoxycholic Acid, Glycyrrhizic Acid, Thymalfasin, Maxamine, VX-497and any compounds that are used to treat HCV through intervention withthe following targets:HCV polymerase, HCV protease, HCV helicase, HCVIRES (internal ribosome entry site).

Non-limiting examples of therapeutic agents for Idiopathic PulmonaryFibrosis with which binding proteins of the invention can be combinedinclude the following: prednisone, azathioprine, albuterol, colchicine,albuterol sulfate, digoxin, gamma interferon, methylprednisolone sodsucc, lorazepam, furosemide, lisinopril, nitroglycerin, spironolactone,cyclophosphamide, ipratropium bromide, actinomycin d, alteplase,fluticasone propionate, levofloxacin, metaproterenol sulfate, morphinesulfate, oxycodone hcl, potassium chloride, triamcinolone acetonide,tacrolimus anhydrous, calcium, interferon-alpha, methotrexate,mycophenolate mofetil, Interferon-gamma-1β.

Non-limiting examples of therapeutic agents for Myocardial Infarctionwith which binding proteins of the invention can be combined include thefollowing: aspirin, nitroglycerin, metoprolol tartrate, enoxaparinsodium, heparin sodium, clopidogrel bisulfate, carvedilol, atenolol,morphine sulfate, metoprolol succinate, warfarin sodium, lisinopril,isosorbide mononitrate, digoxin, furosemide, simvastatin, ramipril,tenecteplase, enalapril maleate, torsemide, retavase, losartanpotassium, quinapril hcl/mag carb, bumetanide, alteplase, enalaprilat,amiodarone hydrochloride, tirofiban hcl m-hydrate, diltiazemhydrochloride, captopril, irbesartan, valsartan, propranololhydrochloride, fosinopril sodium, lidocaine hydrochloride, eptifibatide,cefazolin sodium, atropine sulfate, aminocaproic acid, spironolactone,interferon, sotalol hydrochloride, potassium chloride, docusate sodium,dobutamine hcl, alprazolam, pravastatin sodium, atorvastatin calcium,midazolam hydrochloride, meperidine hydrochloride, isosorbide dinitrate,epinephrine, dopamine hydrochloride, bivalirudin, rosuvastatin,ezetimibe/simvastatin, avasimibe, cariporide.

Non-limiting examples of therapeutic agents for Psoriasis with whichbinding proteins of the invention can be combined include the following:small molecule inhibitor of KDR (ABT-123), small molecule inhibitor ofTie-2, calcipotriene, clobetasol propionate, triamcinolone acetonide,halobetasol propionate, tazarotene, methotrexate, fluocinonide,betamethasone diprop augmented, fluocinolone acetonide, acitretin, tarshampoo, betamethasone valerate, mometasone furoate, ketoconazole,pramoxine/fluocinolone, hydrocortisone valerate, flurandrenolide, urea,betamethasone, clobetasol propionate/emoll, fluticasone propionate,azithromycin, hydrocortisone, moisturizing formula, folic acid,desonide, pimecrolimus, coal tar, diflorasone diacetate, etanerceptfolate, lactic acid, methoxsalen, hc/bismuth subgal/znox/resor,methylprednisolone acetate, prednisone, sunscreen, halcinonide,salicylic acid, anthralin, clocortolone pivalate, coal extract, coaltar/salicylic acid, coal tar/salicylic acid/sulfur, desoximetasone,diazepam, emollient, fluocinonide/emollient, mineral oil/castor oil/nalact, mineral oil/peanut oil, petroleum/isopropyl myristate, psoralen,salicylic acid, soap/tribromsalan, thimerosal/boric acid, celecoxib,infliximab, cyclosporine, alefacept, efalizumab, tacrolimus,pimecrolimus, PUVA, UVB, sulfasalazine.

Non-limiting examples of therapeutic agents for Psoriatic Arthritis withwhich binding proteins of the invention can be combined include thefollowing: methotrexate, etanercept, rofecoxib, celecoxib, folic acid,sulfasalazine, naproxen, leflunomide, methylprednisolone acetate,indomethacin, hydroxychloroquine sulfate, prednisone, sulindac,betamethasone diprop augmented, infliximab, methotrexate, folate,triamcinolone acetonide, diclofenac, dimethylsulfoxide, piroxicam,diclofenac sodium, ketoprofen, meloxicam, methylprednisolone,nabumetone, tolmetin sodium, calcipotriene, cyclosporine, diclofenacsodium/misoprostol, fluocinonide, glucosamine sulfate, gold sodiumthiomalate, hydrocodone bitartrate/apap, ibuprofen, risedronate sodium,sulfadiazine, thioguanine, valdecoxib, alefacept, efalizumab.

Non-limiting examples of therapeutic agents for Restenosis with whichbinding proteins of the invention can be combined include the following:sirolimus, paclitaxel, everolimus, tacrolimus, ABT-578, acetaminophen.

Non-limiting examples of therapeutic agents for Sciatica with whichbinding proteins of the invention can be combined include the following:hydrocodone bitartrate/apap, rofecoxib, cyclobenzaprine hcl,methylprednisolone, naproxen, ibuprofen, oxycodone hcl/acetaminophen,celecoxib, valdecoxib, methylprednisolone acetate, prednisone, codeinephosphate/apap, tramadol hcl/acetaminophen, metaxalone, meloxicam,methocarbamol, lidocaine hydrochloride, diclofenac sodium, gabapentin,dexamethasone, carisoprodol, ketorolac tromethamine, indomethacin,acetaminophen, diazepam, nabumetone, oxycodone hcl, tizanidine hcl,diclofenac sodium/misoprostol, propoxyphene napsylate/apap,asa/oxycod/oxycodone ter, ibuprofen/hydrocodone bit, tramadol hcl,etodolac, propoxyphene hcl, amitriptyline hcl, carisoprodol/codeinephos/asa, morphine sulfate, multivitamins, naproxen sodium, orphenadrinecitrate, temazepam.

Preferred examples of therapeutic agents for SLE (Lupus) in whichbinding proteins of the invention can be combined include the following:NSAIDS, for example, diclofenac, naproxen, ibuprofen, piroxicam,indomethacin; COX2 inhibitors, for example, Celecoxib, rofecoxib,valdecoxib; anti-malarials, for example, hydroxychloroquine; Steroids,for example, prednisone, prednisolone, budenoside, dexamethasone;Cytotoxics, for example, azathioprine, cyclophosphamide, mycophenolatemofetil, methotrexate; inhibitors of PDE4 or purine synthesis inhibitor,for example Cellcept. Binding proteins of the invention, may also becombined with agents such as sulfasalazine, 5-aminosalicylic acid,olsalazine, Imuran and agents which interfere with synthesis, productionor action of proinflammatory cytokines such as IL-1, for example,caspase inhibitors like IL-1β converting enzyme inhibitors and IL-1ra.Binding proteins of the invention may also be used with T cell signalinginhibitors, for example, tyrosine kinase inhibitors; or molecules thattarget T cell activation molecules, for example, CTLA-4-IgG or anti-B7family antibodies, anti-PD-1 family antibodies. Binding proteins of theinvention, can be combined with IL-11 or anti-cytokine antibodies, forexample, fonotolizumab (anti-IFNg antibody), or anti-receptor receptorantibodies, for example, anti-IL-6 receptor antibody and antibodies toB-cell surface molecules. Antibodies of the invention or antigen bindingportion thereof may also be used with LJP 394 (abetimus), agents thatdeplete or inactivate B-cells, for example, Rituximab (anti-CD20antibody), lymphostat-B (anti-BlyS antibody), TNF antagonists, forexample, anti-TNF antibodies, D2E7 (PCT Publication No. WO 97/29131;HUMIRA® adalimumab), CA2 (REMICADE® infliximab), CDP 571, TNFR-Igconstructs, (p75TNFRIgG (ENBREL® etanercept) and p55TNFRIgG(lenercept)).

The pharmaceutical compositions of the invention may include a“therapeutically effective amount” or a “prophylactically effectiveamount” of a binding protein of the invention. A “therapeuticallyeffective amount” refers to an amount effective, at dosages and forperiods of time necessary, to achieve the desired therapeutic result. Atherapeutically effective amount of the binding protein may bedetermined by a person skilled in the art and may vary according tofactors such as the disease state, age, sex, and weight of theindividual, and the ability of the binding protein to elicit a desiredresponse in the individual. A therapeutically effective amount is alsoone in which any toxic or detrimental effects of the antibody, orantibody portion, are outweighed by the therapeutically beneficialeffects. A “prophylactically effective amount” refers to an amounteffective, at dosages and for periods of time necessary, to achieve thedesired prophylactic result. Typically, since a prophylactic dose isused in subjects prior to or at an earlier stage of disease, theprophylactically effective amount will be less than the therapeuticallyeffective amount.

Dosage regimens may be adjusted to provide the optimum desired response(e.g., a therapeutic or prophylactic response). For example, a singlebolus may be administered, several divided doses may be administeredover time or the dose may be proportionally reduced or increased asindicated by the exigencies of the therapeutic situation. It isespecially advantageous to formulate parenteral compositions in dosageunit form for ease of administration and uniformity of dosage. Dosageunit form as used herein refers to physically discrete units suited asunitary dosages for the mammalian subjects to be treated; each unitcontaining a predetermined quantity of active compound calculated toproduce the desired therapeutic effect in association with the requiredpharmaceutical carrier. The specification for the dosage unit forms ofthe invention are dictated by and directly dependent on (a) the uniquecharacteristics of the active compound and the particular therapeutic orprophylactic effect to be achieved, and (b) the limitations inherent inthe art of compounding such an active compound for the treatment ofsensitivity in individuals.

An exemplary, non-limiting range for a therapeutically orprophylactically effective amount of an binding protein of the inventionis 0.1-20 mg/kg, more preferably 1-10 mg/kg. It is to be noted thatdosage values may vary with the type and severity of the condition to bealleviated. It is to be further understood that for any particularsubject, specific dosage regimens should be adjusted over time accordingto the individual need and the professional judgment of the personadministering or supervising the administration of the compositions, andthat dosage ranges set forth herein are exemplary only and are notintended to limit the scope or practice of the claimed composition.

It will be readily apparent to those skilled in the art that othersuitable modifications and adaptations of the methods of the inventiondescribed herein are obvious and may be made using suitable equivalentswithout departing from the scope of the invention or the embodimentsdisclosed herein. Having now described the present invention in detail,the same will be more clearly understood by reference to the followingexamples, which are included for purposes of illustration only and arenot intended to be limiting of the invention.

EXAMPLES Example 1 Generation of Dual Variable Domain Immunoglobulin(DVD-Ig)

The dual variable domain immunoglobulin (DVD-Ig) molecule is designedsuch that two different light chain variable domains (VL) from the twodifferent parent mAbs are linked in tandem directly or via a shortlinker by recombinant DNA techniques, followed by the light chainconstant domain. Similarly, the heavy chain comprises two differentheavy chain variable domains (VH) linked in tandem, followed by theconstant domain CH1 and Fc region (FIG. 1A).

Example 1.1 Generation of Murine Monoclonal Antibodies to IL-1α andIL-1β

Monoclonal Antibodies to IL-1α and IL-1β were generated as follows usingHybridoma technology well known in the art.

Example 1.1.A Immunization of Mice

Purified recombinant human IL-1α and murine IL-1β (R&D Systems) wereused as immunogens as well as coating antigens in titer assays andscreening ELISA. Immunizing dosages ranged from 5.0 to 20.0μg/mouse/injection for all antigens for both primary and boostimmunizations. IMMUNEASY™ adjuvant was purchased from Qiagen (Waltham,Mass.) and used at Adjuvant/antigen ratio of 20 ml IMMUNEASY™ adjuvantper 10.0 μg antigen. Each group of animals to be immunized contained 5IL-1αβ KO mice obtained from Dr. Yoichiro Iwakura (University of Tokyo,Minato-ku, Tokyo, Japan). The mice were immunized according to dosingschedule described below. MRC-5 cells were purchased from ATCC(Manassas, Va.) and used for IL-1 bioassay. Human IL-8 ELISA kits andcontrol mouse anti-hIL-1α and β antibodies (MAB200 and MAB201) werepurchased from R&D Systems (Minneapolis, Minn.).

Briefly, adjuvant-antigen mixture was prepared by first gently mixingthe adjuvant in a vial using a vortex. The desired amount of adjuvantwas removed from the vial and put into an autoclaved 1.5 mLmicrocentrifuge tube. The antigen was prepared in PBS or saline withconcentration ranging from 0.5-1.0 mg/ml. The calculated amount ofantigen was then added to the microcentrifuge tube with the adjuvant andthe solution was mixed by gently pipetting up and down 5 times. Theadjuvant-antigen mixture was incubated at room temperature for 15 minand then mixed again by gently pipetting up and down 5 times. Theadjuvant-antigen solution was drawn into the proper syringe for animalinjection. A total of 5-20 μg of antigen was injected in a volume of50-100 μl. Each animal was immunized, and then boosted 2 to 3 timesdepending on the titer. Animals with good titers were given a finalintravenous boost before fusion and generation of hybridomas.

Example 1.1.B Screening Hybridomas

Hybridomas, generated as described above, were screened and antibodytiter determined using ELISA: Protein antigens were directly coated onELISA plates for detecting the specific antibodies using standard ELISAprocedures. Briefly, ELISA plates were coated with 100 μl of eitherrhIL-1α or rhIL-1β (1.0 μg/ml in PBS) overnight at 4° C. Plates werewashed 3 times with 250 μl PBS/0.5% TWEEN®20 polyethylene-sorbitanmonolaurate and blocked with 200 μl blocking buffer (2% BSA in PBS with0.5% Tween®20 polyethylene-sorbitan monolaurate). Diluted sera orhybridoma supernatant (100 μl) was added to each well, and incubated atroom temperature for 2 hrs. Plates were then washed 3 times withPBS/0.5% Tween®20 polyethylene-sorbitan monolaurate, HRP-goatanti-murine IgG was used for detection, and binding ODs were observed at450 nm. Hybridoma clones producing antibodies that showed high specificbinding activity in the ELISA were subcloned and purified, and affinity(Biacore) and potency (MRC-5 bioassay) of the antibodies werecharacterized as follows.

Example 1.1.C Characterization of Murine Monoclonal Antibodies to IL-1αand IL-1β

The following assays were used to characterize the antibodies producedby the hybridomas described in example 1.1.B.

Example 1.1.C.1 Surface Plasmon Resonance

Real-time binding interactions between captured antibody (mouseanti-rmIL1 antibody captured on a biosensor matrix via goat anti-mouseIgG) and rmIL-1 were measured by surface plasmon resonance (SPR) usingthe BIAcore system (Biacore AB, Uppsala, Sweden) according tomanufacturer's instructions and standard procedures. Briefly, rmIL-1 wasdiluted in HBS running buffer (Biacore AB) and 50 μl aliquots wereinjected through the immobilized protein matrices at a flow rate of 5ml/min. The concentrations of rhIL1 employed were 62.5, 125, 187.5, 250,375, 500, 750, 1000, 1500 and 2000 nM. To determine the dissociationconstant (off-rate), association constant (on-rate), BIAcore kineticevaluation software (version 3.1) was used.

Example 1.1.C.2 Anti-IL-1 Bioassay

The MRC-5 cell line is a human lung fibroblast cell line that producesIL-8 in response to human IL-1α and IL-1β in a dose-dependent manner(see Dinarello, C. A., K. Muegge, and S. K. Durum. 2000. CurrentProtocols in Immunology 6:1). MRC-5 cells were cultured in 10% FBScomplete MEM and grown at 37° C. in a 5% CO₂ incubator. To determineneutralizing potencies of the mAbs against recombinant human IL-1α orIL-1β, different concentrations (0-10 μg/ml) of mAb (50 μl) was added toa 96-well plate and pre-incubated with 50 μl of rhIL-1a or rhIL-1b(10-50 pg/ml) for 1 hr at 37° C. The supernatants were harvested,diluted, and IL-8 concentrations measured by ELISA using a standard IL-8ELISA kit (R&D Systems). Antibody potency was determined by its abilityto inhibit IL-8 production by MRC-5 cells.

Based on Biacore and MRC-5 bioassay, a number of murine anti-hIL-1a andanti-hIL-1b antibodies with high affinity and potency were identified,as shown in Table 1 below:

TABLE 1 Generation and characterization of murine anti-hIL-1a/b mAbs.mAb Clone# Specificity K_(D) (M) IC₅₀ (M) 3D12.E3 hIL-1α 1.11E−096.70E−10 18F4.2C8 hIL-1α 5.78E−10 8.90E−11 6H3.1A4.3E11 hIL-1α 3.54E−102.40E−10 13F5.G5 hIL-1β 2.91E−10 6.00E−10 1B12.4H4 hIL-1β 2.13E−105.30E−10 6B12.4F6 hIL-1β 5.54E−10 3.20E−10

Example 1.1.D Cloning and Sequencing of the Murine Monoclonal Antibodiesto IL-1α and IL-β

Cloning and sequencing of the variable heavy (VH) and light (VL) genesof all anti-IL-1a/b mAbs described in Table 1 and additional antibodieswere carried out after isolation and purification of the total RNA fromthe each hybridoma cell line using TRIZOL® reagent (Invitrogen)according to the manufacturer's instructions. Amplification of both VHand VL genes was carried out using the IgGVH and IgκVL oligonucleotidesfrom the Mouse Ig-Primer Set (Novagen, Madison, Wis.) with One-tubeRT-PCR kit (Qiagen) as suggested by the manufacturer. DNA fragmentsresulting from productive amplifications were cloned into pCR-TOPO®vector (Invitrogen) according to the manufacturer's instructions.Multiple VH and VL clones were then sequenced by the dideoxy chaintermination method using an ABI 3000 sequencer (Applied Biosystems,Foster City, Calif.). The sequences of all mAb VL and VH genes are shownbelow in Table 2.

TABLE 2 Murine monoclonal antibodies capable of binding human IL-1αor IL-1β Sequence Sequence Protein Identifier 12345678901234567890VH 3D12.E3 SEQ ID NO.: 1 QIQLVQSGPELKKPGETVKI SCKASGYTFRNYGMNWVKQAPGKDLKRMAWINTYTGESTY ADDFKGRFAFSLETSASTAY LQINNLKNEDTATYFCARGIYYYGSSYAMDYWGQGTSVTV SS VL 3D12.E3 SEQ ID NO.: 2 NIQMTQTTSSLSASLGDRVTISCRASQDISNCLNWYQQKP DGTVKLLIYYTSRLHSGVPS RFSGSGSGTDYSLTISNLEQEDIATYFCQQGKTLPYAFGG GTKLEINR VH 18F4.2C8 SEQ ID NO.: 3EVQLQQSGAELVKPGASVKL SCTASGLNIKDTYMHWLKQR PEQGLEWIGRIDPANGNAKYDPRFLGKATITADTSSNTAY LQLSSLTSEDTAVYYCARGD GNFHFDYWGQGTTLTVSS VL 18F4.2C8SEQ ID NO.: 4 DIVMTQSQRFMSTSVGDRVS VTCKASQNVGTNIAWYQQKPGQSPRALIYSASYRYSGVPD RFTGSGSGTDFTLTISNVQS VDLAEYFCQQYTRYPLTFGG GTKLEIKRVH SEQ ID NO.: 5 QVQLQQPGAELVRPGASVKL 6H3.1A4.3E11 SCKASGYTFTTYWMNWVKQRPEQGLEWIGRIDPYDSETLY SQKFKDTAILTVDKSSSTAY MQLSSLTSEDSAVYYCARYGFDYWGQGTTLTVSS VL SEQ ID NO.: 6 QIVLTQSPALMSASPGEKVT 6H3.1A4.3E11MTCSASSSVNYMYWYQQKPR SSPKPWIYLTSNLASGVPAR FSGSGSGTSYSLTISSMEAEDAATYYCQQWNSNPYTFGGG TKLEMKR VH 13F5.G5 SEQ ID NO.: 7QVQLQQSGAELVRPGSSVKI SCKASGYAFSSYWMNWVKQR PGQGLEWIGQIYPGDGDTNYNGKFKGKATLTADKSSSTSY MQLSGLTSEDSAMYFCVRFP TGNDYYAMDYWGQGTSVTVSSVL 13F5.G5 SEQ ID NO.: 8 NIVLTQSPASLAVSLGQRAT ISCRASESVDSYGNSYMHWYQQKPGQPPKLLIYLASNLES GVPARFSGSGSRTDFTLTID PVEADDAATYYCQQNNEDPFTFGSGTKLEIKR VH 1B12.4H4 SEQ ID NO.: 9 QVHLKESGPGLVAPSQSLSITCTVSGFSLTDYGVSWIRQP PGKGLEWLGLIWGGGDTYYN SPLKSRLSIRKDNSKSQVFLKMNSLQTDDTAVYYCAKQRT LWGYDLYGMDYWGQGTSVTV SS VL 1B12.4H4 SEQ ID NO.: 10ETTVTQSPASLSMAIGEKVT IRCITSTDIDVDMNWYQQKP GEPPKLLISQGNTLRPGVPSRFSSSGSGTDFVFIIENMLS EDVADYYCLQSDNLPLTFGA GTKLELKR VH 6B12.4F6SEQ ID NO.: 11 EVQLQQSGPELVKTGTSVKI SCKASGYSFTGYYMHWVRQSHGKSLEWIGYISCYNGFTSY NPKFKGKATFTVDTSSSTAY IQFSRLTSEDSAVYYCARSDYYGTNDYWGQGTTLTVSS VL 6B12.4F6 SEQ ID NO.: 12 QIVLTQSPAIMSASPGEKVTITCSASSSVSYMHWFQQKPG ASPKLWIYSTSNLASGVPAR FSGSGSGTSYSLTVSRMEAEDAATYYCQQRSTYPYTFGGG TKLEIKR

Example 1.2 Generation and Characterization of Murine-Human ChimericAntibodies

All mAbs described above were converted to chimeric (with human constantregion) and expressed, purified, and characterized to confirm activityand will be used as controls for subsequent DVD-Ig analysis. To convert3D12.E3 into chimeric form, 3D12.E3-VL was PCR amplified using primersP1 and P2; meanwhile human Ck gene (in pBOS vector generated in-house atABC) was amplified using primers P3 and P4. Both PCR reactions wereperformed according to standard PCR techniques and procedures. The twoPCR products were gel-purified, and used together as overlappingtemplate for the subsequent overlapping PCR reaction using primers P1and P4 using standard PCR conditions. The final PCR product, thechimeric light chain 3D12.E3-VL-hCk, was subcloned into pEF6 TOPO®mammalian expression vector (Invitrogen) by TOPO® vector cloningaccording to the manufacturer's instructions. Table 3 shows the PCRprimers' sequences:

TABLE 3 P1: 5′ ATG GTG TCC ACA GCT CAG SEQ ID NO. 13 TTC C 3′ P2: 5′GC AGC CAC CGT ACG CCG GTT TAT SEQ ID NO. 14 TTC CAG 3′ P3: 5′CGT ACG GTG GCT GCA CCA TCT SEQ ID NO. 15 GTC 3′ P4: 5′TCA ACA CTC TCC CCT GTT SEQ ID NO. 16 GAA GC 3′

To convert 3D12.E3 heavy chain into chimeric form, 3D12.E3-VH was PCRamplified using primers P5 and P6; meanwhile human Cγ1 gene (in pBOSvector generated in-house at ABC) was amplified using primers P7 and P8.Both PCR reactions were performed according to standard PCR techniquesand procedures. The two PCR products were gel-purified, and usedtogether as overlapping template for the subsequent overlapping PCRreaction using primers P5 and P8 using standard PCR conditions. Thefinal PCR product, the chimeric light chain 3D12.E3-VH-hCγ1, wassubcloned into pcDNA3.1 TOPO® mammalian expression vector (Invitrogen)according to the manufacturer's instructions. Table 4 shows the PCRprimers' sequences:

TABLE 4 P5: 5′ ATG GCT TGG GTG TGG ACC SEQ ID NO. 17 TTG C 3′ P6: 5′GGG CCC TTG GTC GAC GCT GAG SEQ ID NO. 18 GAG ACG GTG ACT GAG G 3′P7: 5′ GCG TCG ACC AAG GGC CCA TCG SEQ ID NO. 19 GTC TTC C 3′ P8: 5′TC ATT TAC CCG GAG ACA GGG AGA SEQ ID NO. 20 GGC 3′

Similarly, chimeric 13F5.G5-VH-Cγ1 was generated using primers P21/P22(for VH) and P7/P8 (for hCγ1) and cloned into pcDNA3.1 TOPO® vecter, andchimeric 13F5.G5-VL-Cκ was generated using primers P23/P24 (for VL) andP3/P4 (for hCk) and cloned into pEF6 TOPO® vector. Table 5 shows the PCRprimers' sequences:

TABLE 5 P21: 5′ ATA GAA TGG AGC TGG GTT TTC SEQ ID NO. 21 CTC 3′ P22: 5′GGG CCC TTG GTC GAC GC TGA SEQ ID NO. 22 GGA GAC GGT GAC TGA 3′ P23: 5′ATG GTC CTC ATG TCC TTG CTG SEQ ID NO. 23 TTC 3′ P24: 5′GC AGC CAC CGT ACG CCG TTT SEQ ID NO. 24 TAT TTC CAG CTT TG 3′

To express chimeric Abs, 13F5.G5-VL-Cκ and 13F5.G5-VH-Cγ1 wereco-expressed in COS using LIPOFECTAMINE™ transfection reagent(Invitrogen) for 72 hr, and the medium collected and IgG purified byProtein A chromatography. Similarly, 13F5.G5-VL-Cκ and 13F5.G5-VH-Cγ1were co-expressed in COS using LIPOFECTAMINE™ transfection reagent(Invitrogen) for 72 hr, and the medium collected and IgG purified byProtein A chromatography. Both purified chimeric Abs were characterizedby Biacore and MRC-5 bioassay to confirm activity. The results showedthat these chimeric Abs displayed similar affinity and potency to thatof the original murine mAbs.

Example 1.3 Construction, Expression, and Purification of IL-1α/β DualVariable Domain Immunoglobulin (DVD-Ig)

The construct used to generate DVD-Ig capable of binding hIL-1α andhIL-1β is illustrated in FIG. 1B. Briefly, parent mAbs including twohigh affinity murine Abs, anti-hIL-1α (clone 3D12.E3) and anti-hIL-1β(clone 13F5.G5), were obtained by immunizing Balb/c mice withrecombinant IL-1α protein (rhIL-1α) and recombinant IL-1β protein(rhIL-1β), respectively. The VL/VH genes of these two hybridoma cloneswere isolated by RT-PCR using the mouse Ig Primer Kit (Novagen, Madison,Wis.). The VL/VH genes were first converted into chimeric antibodies(with human constant regions) to confirm activity and potency. Togenerate DVD1-Ig, the VH and VL of 13F5.G5 was directly fused to theN-terminus of the VH and VL of 3D12.E3, respectively (as shown in FIG.1B). The DVD2-Ig was constructed similarly, except that it had a linkerbetween the two variable domains in both the light chain (the linkersequence is ADAAP (SEQ ID NO:40)) and the heavy chain (the linkersequence is AKTTPP (SEQ ID NO:38)). These sequences were selected fromthe N-termini of murine Ck and CH1 sequences. These linker sequences,selected from the N-termini of murine Ck and CH1, are natural extensionof the variable domains and exhibit a flexible conformation withoutsignificant secondary structures based on the analysis of several Fabcrystal structures. The detailed procedures of the PCR cloning isdescribed below:

Example 1.3.A Molecular Cloning of hIL-1a/bDVD1-Ig

13F5.G5-VH was PCR amplified using primers P21 and P25; meanwhile3D12.E3-VH-hCγ1 was amplified using primers P14 and P8. Both PCRreactions were performed according to standard PCR techniques andprocedures. The two PCR products were gel-purified, and used together asoverlapping template for the subsequent overlapping PCR reaction usingprimers P21 and P8 using standard PCR conditions. The final PCR product,the DVD1-Ig heavy chain hIL-1a/bDVD1-VH-hCγ1, was subcloned intopcDNA3.1 TOPO® mammalian expression vector (Invitrogen) according to themanufacturer's instructions. Table 6 shows the PCR primers' sequences:

TABLE 6 P14: 5′ CAG ATC CAG TTG GTG CAG TCT SEQ ID NO. 25 GG3′ P25: 5′CAC CAA CTG GAT CTG TGA GGA SEQ ID NO. 26 GAC GGT GAC TGA GG 3′

To generate hIL-1a/bDVD1-Ig light chain, 13F5.G5-VL was PCR amplifiedusing primers P23 and P26; meanwhile 3D12.E3-VL-hCκ was amplified usingprimers P16 and P4. Both PCR reactions were performed according tostandard PCR techniques and procedures. The two PCR products weregel-purified, and used together as overlapping template for thesubsequent overlapping PCR reaction using primers P23 and P4 usingstandard PCR conditions. The final PCR product, the hIL-1a/bDVD1-Iglight chain hIL-1a/bDVD1-VL-hCκ, was subcloned into pEF6 TOPO® mammalianexpression vector (Invitrogen) according to the manufacturer'sinstructions. Table 7 shows the PCR primers' sequences:

TABLE 7 P16: 5′ AAT ATC CAG ATG ACA CAG ACT SEQ ID NO. 27 ACA TCC 3′P26: 5′ GTGT CAT CTG GAT ATT CCG TTT SEQ ID NO. 28 TAT TTC CAG CTT TG 3′

Example 1.3.B Molecular Cloning of hIL-1a/bDVD2-Ig

13F5.G5-VH was PCR amplified using primers P21 and P17; meanwhile3D12.E3-VH-hCγ1 was amplified using primers P18 and P8. Both PCRreactions were performed according to standard PCR techniques andprocedures. The two PCR products were gel-purified, and used together asoverlapping template for the subsequent overlapping PCR reaction usingprimers P21 and P8 using standard PCR conditions. The final PCR product,the DVD2-Ig heavy chain hIL-1a/bDVD2-VH-hCγ1, was subcloned intopcDNA3.1 TOPO® mammalian expression vector (Invitrogen) according to themanufacturer's instructions. Table 8 shows the PCR primers' sequences:

TABLE 8 P17: 5′ TGG GGG TGT CGT TTT GGC TGA SEQ ID NO. 29 GG 3′ P18: 5′GCC AAA ACG ACA CCC CCA CAG SEQ ID NO. 30 ATC CAG TTG GTG CAG 3′

To generate hIL-1a/bDVD2-Ig light chain, 13F5.G5-VL was PCR amplifiedusing primers P23 and P19; meanwhile 3D12.E3-VL-hCκ was amplified usingprimers P20 and P4. Both PCR reactions were performed according tostandard PCR techniques and procedures. The two PCR products weregel-purified, and used together as overlapping template for thesubsequent overlapping PCR reaction using primers P23 and P4 usingstandard PCR conditions. The final PCR product, the hIL-1a/bDVD2-Iglight chain hIL-1a/bDVD2-VL-hCκ, was subcloned into pEF6 TOPO® mammalianexpression vector (Invitrogen) according to the manufacturer'sinstructions. Table 9 shows the PCR primers' sequences:

TABLE 9 P19: 5′ TGG TGC AGC ATC AGC CCG TTT SEQ ID NO. 31 TAT TTC 3′P20: 5′ GCT GAT GCT GCA CCA AAT ATC SEQ ID NO. 32 CAG ATG ACA CAG 3′

The final sequences of hIL-1a/bDVD1-Ig and hIL-1a/bDVD2-Ig are describedin Table 10:

TABLE 10 Amino acid sequence of hIL-1α/βDVD1-Ig and hIL-1α/βDVD2-IgProtein Sequence Sequence Protein region Identifier 12345678901234567890DVD HEAVY SEQ ID NO.: 33 QVQLQQSGAELVRPGSSVKI VARIABLESCKASGYAFSSYWMNWVKQR hIL-1a/bDVD1- PGQGLEWIGQIYPGDGDTNY IgNGKFKGKATLTADKSSSTSY MQLSGLTSEDSAMYFCVRFP TGNDYYAMDYWGQGTSVTVSSQIQLVQSGPELKKPGETVK ISCKASGYTFRNYGMNWVKQ APGKDLKRMAWINTYTGESTYADDFKGRFAFSLETSASTA YLQINNLKNEDTATYFCARG IYYYGSSYAMDYWGQGTSVT VSSVH 13F5.G5 SEQ ID NO.: 7 QVQLQQSGAELVRPGSSVKI SCKASGYAFSSYWMNWVKQRPGQGLEWIGQIYPGDG DTNYNGKFKGKATLTADKSS STSYMQLSGLTSEDSAMYFCVRFPTGNDYYAMDYWG QGTSVTVSS Linker None 3D12.E3 VH SEQ ID NO.: 1QIQLVQSGPELKKPGETVKI SCKASGYTFRNYGMNWVKQA PGKDLKRMAWINTYTGESTYADDFKGRFAFSLETSASTAY LQINNLKNEDTATYFCARGI YYYGSSYAMDYWGQGTSVTV SS CHSEQ ID NO.: 34 ASTKGPSVFPLAPSSKSTSG GTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSS GLYSLSSVVTVPSSSLGTQT YICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGG PSVFLFPPKPKDTLMISRTP EVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYN STYRVVSVLTVLHQDWLNGK EYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREE MTKNQVSLTCLVKGFYPSDI AVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRW QQGNVFSCSVMHEALHNHYT QKSLSLSPGK DVD LIGHTSEQ ID NO.: 35 NIVLTQSPASLAVSLGQRAT VARIABLE ISCRASESVDSYGNSYMHWYhIL-1a/bDVD1- QQKPGQPPKLLIYLASNLES Ig GVPARFSGSGSRTDFTLTIDPVEADDAATYYCQQNNEDPF TFGSGTKLEIKRNIQMTQTT SSLSASLGDRVTISCRASQDISNCLNWYQQKPDGTVKLLI YYTSRLHSGVPSRFSGSGSG TDYSLTISNLEQEDIATYFCQQGKTLPYAFGGGTKLEINRR 13F5.G5 VL SEQ ID NO.: 8 NIVLTQSPASLAVSLGQRATISCRASESVDSYGNSYMHWY QQKPGQPPKLLIYLASNLES GVPARFSGSGSRTDFTLTIDPVEADDAATYYCQQNNEDPF TFGSGTKLEIKR Linker None 3D12.E3 VL SEQ ID NO.: 2NIQMTQTTSSLSASLGDRVT ISCRASQDISNCLNWYQQKP DGTVKLLIYYTSRLHSGVPSRFSGSGSGTDYSLTISNLEQ EDIATYFCQQGKTLPYAFGG GTKLEINR CL SEQ ID NO.: 36TVAAPSVFIFPPSDEQLKSG TASVVCLLNNFYPREAKVQW KVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEK HKVYACEVTHQGLSSPVTKS FNRGEC DVD HEAVYSEQ ID NO.: 37 QVQLQQSGAELVRPGSSVKI VARIABLE SCKASGYAFSSYWMNWVKQRhIL-1a/bDVD2- PGQGLEWIGQIYPGDGDTNY Ig NGKFKGKATLTADKSSSTSYMQLSGLTSEDSAMYFCVRFP TGNDYYAMDYWGQGTSVTVS SAKTTPPQIQLVQSGPELKKPGETVKISCKASGYTFRNYG MNWVKQAPGKDLKRMAWINT YTGESTYADDFKGRFAFSLETSASTAYLQINNLKNEDTAT YFCARGIYYYGSSYAMDYWG QGTSVTVSS 13F5.G5 VHSEQ ID NO.: 7 QVQLQQSGAELVRPGSSVKI SCKASGYAFSSYWMNWVKQRPGQGLEWIGQIYPGDGDTNY NGKFKGKATLTADKSSSTSY MQLSGLTSEDSAMYFCVRFPTGNDYYAMDYWGQGTSVTVSS Linker SEQ ID NO.: 38 AKTTPP 3D12.E3 VHSEQ ID NO.: 1 QIQLVQSGPELKKPGETVKI SCKASGYTFRNYGMNWVKQAPGKDLKRMAWINTYTGESTY ADDFKGRFAFSLETSASTAY LQINNLKNEDTATYFCARGIYYYGSSYAMDYWGQGTSVTV SS CH SEQ ID NO.: 34 ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVS WNSGALTSGVHTFPAVLQSS GLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEP KSCDKTHTCPPCPAPELLGG PSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNW YVDGVEVHNAKTKPREEQYN STYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTIS KAKGQPREPQVYTLPPSREE MTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPV LDSDGSFFLYSKLTVDKSRW QQGNVFSCSVMHEALHNHYTQKSLSLSPGK DVD LIGHT SEQ ID NO.: 39 NIVLTQSPASLAVSLGQRAT VARIABLE HIL-ISCRASESVDSYGNSYMHWY 1a/bDVD2- QQKPGQPPKLLIYLASNLES IgGVPARFSGSGSRTDFTLTID PVEADDAATYYCQQNNEDPF TFGSGTKLEIKRADAAPNIQMTQTTSSLSASLGDRVTISC RASQDISNCLNWYQQKPDGT VKLLIYYTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDI ATYFCQQGKTLPYAFGGGTK LEINR 13F5.G5 VL SEQ ID NO.: 8NIVLTQSPASLAVSLGQRAT ISCRASESVDSYGNSYMHWY QQKPGQPPKLLIYLASNLESGVPARFSGSGSRTDFTLTID PVEADDAATYYCQQNNEDPF TFGSGTKLEIKR LinkerSEQ ID NO.: 40 ADAAP 3D12.E3 VL SEQ ID NO.: 2 NIQMTQTTSSLSASLGDRVTISCRASQDISNCLNWYQQKP DGTVKLLIYYTSRLHSGVPS RFSGSGSGTDYSLTISNLEQEDIATYFCQQGKTLPYAFGG GTKLEINR CL SEQ ID NO.: 36 TVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQW KVDNALQSGNSQESVTEQDS KDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKS FNRGEC

Example 1.3.C Expression and Purification of hIL-1a/bDVD1-Igs

The heavy and light chain of each construct was subcloned into pcDNA3.1TOPO® and pEF6 TOPO® vectors (Invitrogen Inc.), respectively, andsequenced to ensure accuracy. The plasmids encoding the heavy and lightchains of each construct were transiently expressed using LIPOFECTAMINE™2000 and 293Fectin™ transfection reagents, respectively in COS cells aswell as human embryonic kidney 293 cells (American Type CultureCollection, Manassas, Va.). The cell culture media was harvested 72hr-post transient transfection and antibodies purified using protein Achromatography (Pierce, Rockford, Ill.) according to manufacturer'sinstructions. The Abs were analyzed by SDS-PAGE and quantitated by A280and BCA (Pierce, Rockford, Ill.). Table 11 shows that the expressionlevels of hIL-1a/bDVD1-Ig and hIL-1a/bDVD2-Ig are comparable to that ofthe chimeric Abs, indicating that the DVD-Ig can be expressedefficiently in mammalian cells.

TABLE 11 Expression and molecular weight analysis of hIL-1a/bDVD-IgExpression level Molecular mass (ng/ml) (Dalton) COS Freestyle 293 LightHeavy Mock 0 0 Chain Chain Full length 3D12.E3-Ch 2788 3886 23,69649,914 147,220 13F5.G5-Ch 3260 3562 24,084 49,518 147,204 DVD1-Ig 29883300 35,797 64,380 200,346 (35,790) (64,371) (200,521) DVD2-Ig 2433 348636,222 64,976 202,354 (36,220) (64,973) (202,573) The molecular mass ofthe light chain, heavy chain, and full length of DVD1-Ig and DVD2-Igdetermined experimentally by mass spectrometry are shown in parenthesis.

Example 1.4 Mass Spectrometry and SEC Analysis of hIL-1a/b DVD-Ig

For measuring molecular weight (MW) of light and heavy chains of DVD-Ig,10 uL of DVD-Ig (0.8 ug/uL) was reduced by 1.0 M DTT solution (5 uL). APLRP-S, 8 u, 4000 A, and 1×150 mm protein column (Michrom BioResource,Auburn, Mass.) was used to separate heavy and light chains of DVD-Ig.Agilent HP1100 Capillary HPLC (Agilent Technologies Inc., Pala Alto,Calif.) was used with the mass spectrometer QSTAR® (Applied Biosystems,Foster City, Calif.). The valco valve was set at 10 minutes to switchthe flow from waste to MS for desalting sample. Buffer A was 0.02% TFA,0.08% FA, 0.1% ACN and 99.8% HPLC-H2O. Buffer B contained 0.02% TFA,0.08% FA, 0.1% HPLC-H2O, and 99.8% ACN. The HPLC flow rate was 50uL/min, and the sample injection volume was 8.0 mL. The temperature ofthe column oven was set at 60° C., and separation gradient was: 5% B for5 minutes; 5% B to 65% B for 35 minutes; 65% B to 95% B for another 5minutes, and 95% B to 5% B for 5 minutes. TOFMS scan was from 800 to2500 amu, and cycles were 3600. To determine the MW of full lengthDVD-Ig, a Protein MICROTRAP™ cartridge (Michrom BioResource, Auburn,Mass.) was used for desalting the sample. The HPLC gradient was: 5% Bfor 5 minutes; 5% B to 95% B in 1 minutes; and from 95% B to 5% B inanother 4 minutes. The QSTAR® TOFMS scan was from 2000 to 3500 amu, andcycles were 899. All MS raw data were analyzed using the Analyst QSsoftware (Applied Biosystems). For SEC analysis of the DVD-Ig, purifiedDVD-Ig and chimeric Abs, in PBS, were applied on a Superose 6 10/300 G2,300×10 mm column (Amersham Bioscience, Piscataway, N.J.). An HPLCinstrument, Model 10A (Shimadzu, Columbia, Md.) was used for SEC. Allproteins were determined using UV detection at 280 nm and 214 nm. Theelution was isocratic at a flow rate of 0.5 mL/min. For stability study,samples in the concentration range of 0.2-0.4 mg/ml in PBS underwent 3freeze-thaw cycles between −80° C. and 25° C., or were incubated at 4°C., 25° C., or 40° C., for 4 weeks and 8 weeks, followed by SECanalysis.

DVD-Ig and chimeric Abs were purified by protein A chromatography. Thepurification yield (3-5 mg/L) was consistent with hIgG quantification ofthe expression medium for each protein. The composition and purity ofthe purified DVD-Igs and chimeric Abs were analyzed by SDS-PAGE in bothreduced and non-reduced conditions. In non-reduced condition, each ofthe four proteins migrated as a single band. The DVD-Ig proteins showedlarger M.W. than the chimeric Abs, as expected. In non-reducingcondition, each of the four proteins yielded two bands, one heavy chainand one light chain. Again, the heavy and light chains of the DVD-Igswere larger in size than that of the chimeric Abs. The SDS-PAGE showedthat each DVD-Ig is expressed as a single species, and the heavy andlight chains are efficiently paired to form an IgG-like molecule. Thesizes of the heavy and light chains as well as the full-length proteinof two DVD-Ig molecules are consistent with their calculated molecularmass based on amino acid sequences (see Table 11).

In order to determine the precise molecular weight of DVD-Ig, massspectrometry was employed. As shown in Table I, the experimentallydetermined molecular mass of each DVD-Ig, including the light chain,heavy chain, and the full-length protein, is in good agreement with thepredicted value. To further study the physical properties of DVD-Ig insolution, size exclusion chromatography (SEC) was used to analyze eachprotein. Both chimeric Abs and DVD2-Ig exhibited a single peak,demonstrating physical homogeneity as monomeric proteins. The 3D12.E3chimeric Ab showed a smaller physical size then 13F5.G5 chimeric Ab,indicating that 3D12.E3 chimeric Ab adopted a more compact, globularshape. DVD1-Ig revealed a major peak as well as a shoulder peak on theright, suggesting that a portion of DVD1-Ig is possibly in an aggregatedform in current buffer condition.

Example 1.5 Analysis of In Vitro Stability of hIL-1a/b DVD-Igs

The physical stability of DVD-Ig was tested as follows. Purifiedantibodies in the concentration range of 0.2-0.4 mg/ml in PBS underwent3 freeze-thaw cycles between −80° C. and 25° C., or were incubated at 4°C., 25° C., or 40° C., for 4 weeks and 8 weeks, followed by analysisusing size exclusion chromatography (SEC) analysis (see Table 12).

TABLE 12 in vitro stability analysis of hIL-1a/b DVD-Ig by SEC D12.E3-Ch3F5.G5-Ch DVD1-Ig DVD2-Ig Agg Ab Frgm Agg Ab Frgm Agg Ab Frgm Agg AbFrgm 3xFreeze- 1.72 98.28 0.00 13.0 87.0 0.0 46.50 53.50 0.00 0.0 100.00.0 Thaw  4° C. @ 4 Wks 0.85 99.15 0.00 4.2 95.8 0.0 42.43 56.63 0.940.0 100.0 0.0 25° C. @ 4 Wks 1.29 98.71 0.00 0.0 100.0 0.0 45.66 54.340.00 0.0 100.0 0.0 40° C. @ 4 Wks 1.65 98.35 0.00 20.3 78.1 1.6 36.7059.42 3.88 0.0 100.0 0.0  4° C. @ 8 Wks 5.35 90.33 4.32 2.2 97.8 0.038.18 56.91 4.91 0.0 100.0 0.0 25° C. @ 8 Wks 1.11 60.55 38.34 1.4 97.51.0 24.42 67.39 8.19 0.0 100.0 0.0 40° C. @ 8 Wks 4.74 81.47 13.79 34.665.4 0.0 20.55 67.16 12.29 0.0 100.0 0.0 The degree of aggregation andfragmentation are shown in percentage, whereas the percentage of Abrepresents intact molecule. Agg: aggregates; Ab: intact antibody; Frgm:fragments.

Both chimeric Abs showed minor degrees of aggregation and fragmentation,normal for a regular IgG molecule. DVD1-Ig showed some aggregation onSCE after purification. In the stability analysis, DVD1-Ig also showedaggregations in PBS under different conditions; however the percentageof aggregated form of DVD1-Ig did not increase during prolonged storageor at higher temperatures. The percentage of the fragmented form ofDVD1-Ig were in the normal range, similar to that of the chimeric3D12.E3 Ab. In contrast, DVD2-Ig showed exceptional stability. Neitheraggregation nor fragmentation was detected for DVD2-Ig in all conditionstested, and 100% of DVD2-Ig maintained as intact monomeric molecule.

Example 1.6 Determination of Antigen Binding Affinity of hIL-1a/bDVD-Igs

The kinetics of DVD-Ig binding to rhIL1-α and rhIL1-β was determined bysurface plasmon resonance-based measurements with a Biacore 3000instrument (Biacore AB, Uppsala, Sweden) using HBS-EP (10 mM HEPES, pH7.4, 150 mM NaCl, 3 mM EDTA, and 0.005% surfactant P20) at 25° C. Allchemicals were obtained from Biacore AB (Uppsala, Sweden) or otherwisefrom a different source as described herein. Approximately, 5000 RU ofgoat anti-human IgG Fcγ fragment specific polyclonal antibody (PierceBiotechnology Inc, Rockford, Ill.) diluted in 10 mM sodium acetate (pH4.5) was directly immobilized across a CM5 research grade biosensor chipusing a standard amine coupling kit according to manufacturer'sinstructions and procedures at 25 mg/ml. Unreacted moieties on thebiosensor surface were blocked with ethanolamine. Modified carboxymethyldextran surface in flowcell 2 and 4 was used as a reaction surface.Unmodified carboxymethyl dextran without goat anti-human IgG in flowcell 1 and 3 was used as the reference surface. For kinetic analysis,rate equations derived from the 1:1 Langmuir binding model were fittedsimultaneously to association and dissociation phases of all teninjections (using global fit analysis) using the Bioevaluation 4.0.1software. Purified DVD-Ig samples were diluted in HEPES-buffered salinefor capture across goat anti-human IgG Fc specific reaction surfaces andinjected over reaction matrices at a flow rate of 5 ml/min. Theassociation and dissociation rate constants, kon (M−1 s−1) and koff(s−1) were determined under a continuous flow rate of 25 ml/min. Rateconstants were derived by making kinetic binding measurements at tendifferent antigen concentrations ranging from 1.25 to 1000 nM. Theequilibrium dissociation constant (M) of the reaction between DVD-Ig andhIL1α/β was then calculated from the kinetic rate constants by thefollowing formula: KD=koff/kon. Aliquots of rhIL1α/β samples were alsosimultaneously injected over a blank reference and reaction CM surfaceto record and subtract any nonspecific binding background to eliminatethe majority of the refractive index change and injection noise.Surfaces were regenerated with two subsequent 25 ml injections of 10 mMGlycine (pH 1.5) at a flow rate of 5 ml/min. The anti-Fc antibodyimmobilized surfaces were completely regenerated and retained their fullcapture capacity over twelve cycles. The apparent stoichiometry of thecaptured DVD-Ig-rhIL1α/β complex was calculated under saturating bindingconditions (steady-state equilibrium) using the following formula:

${Stoichiometry} = {\frac{{rhIL}\; 1{\alpha/\beta}\mspace{20mu}{response}\mspace{14mu}({RU})}{{DVD}\mspace{14mu}{response}\mspace{14mu}({RU})} \times \frac{ {{DVD}\text{-}{Ig}\mspace{14mu}{MW}} )}{{rhIL}\; 1{\alpha/\beta}\mspace{14mu}{MW}}}$

The Biacore analysis indicated the chimeric Abs possessed similarbinding kinetics and affinities to IL-1 as the original hybridoma mAbs,indicating that the correct VL/VH sequences had been isolated (TableIII). The overall binding parameters of the two DVD-Igs to hIL-1α weresimilar, with the affinities of the DVD-Igs being only 2-3 fold lessthan that of the chimeric 3D12.E3 Ab. The binding affinity of DVD2-Ig tohIL-1β was slightly less than the chimeric Ab 13F5.G5, but 3-fold higherthan that of DVD1-Ig. The affinity of the two DVD-Igs to hIL-1 ascompared to the affinity of chimeric Abs to hIL-1 was similar asindicated by the evaluation of the stoichiometry to IL-1. Both chimericAbs, being bivalent monospecific, bound to IL-1α and IL-1β on Biocorewith a stoichiometry of 1.6 and 1.7, respectively. This is common for anIgG due to inter-molecular interference when antibodies are immobilizeddensely on the Biacore sense chip resulting in stoichiometry being inthe range from 1.5 to 2.0. The stoichiometry of both DVD-Igs for hIL-1αand hIL-1β were similar to that of the two chimeric Abs, indicating thatboth DVD-Igs possessed bivalent binding capability to each antigen.

TABLE 13 Functional characterization of anti-IL-1 DVD-Ig molecule k_(on)k_(off) K_(d) Potency Antigen (M−1 s−1) (s−1) (M) Stoichiometry IC₅₀ (M)3D13.E3 hIL-1α 6.43E+05 7.13E−04 1.11E−09 2.0 6.70E−10 3D12.E3-Ch hIL-1α4.12E+05 5.52E−04 1.34E−09 1.6 7.00E−10 DVD1-Ig hIL-1α 3.70E+04 1.05E−042.83E−09 1.8 2.30E−09 DVD2-Ig hIL-1α 7.35E+04 2.52E−04 3.42E−09 2.02.90E−09 13F5.G5 hIL-1β 2.13E+06 6.21E−04 2.91E−10 1.8 6.00E−1013F5.G5-Ch hIL-1β 1.41E+06 6.54E−04 4.62E−10 1.7 5.30E−10 DVD1-Ig hIL-1β6.09E+05 1.59E−03 2.60E−09 1.5 3.10E−09 DVD2-Ig hIL-1β 1.19E+06 9.50E−047.98E−10 1.8 1.60E−09 Affinity and stoichiometry were measured byBiacore; Potency (IC₅₀) was determined by MRC-5 bioassay.

In addition, tetravalent dual-specific antigen binding of DVD-Ig wasalso analyzed by Biacore (Table 14). DVD-Ig was first captured via agoat anti-human Fc antibody on the Biacore sensor chip, and the firstantigen was injected and a binding signal observed. As the DVD-Ig wassaturated by the first antigen, the second antigen was then injected andthe second signal observed. This was done either by first injectingIL-1β then IL-1α or by first injecting IL-1α followed by IL-1β forDVD2-Ig. In either sequence, a dual-binding activity was detected.Similar results were obtained for DVD1-Ig. Thus each DVD-Ig was able tobind both antigens simultaneously as a dual-specific tetravalentmolecule. As shown in Table IV, the stoichiometry of both DVD-Ig to thefirst antigen, either hIL-1α or hIL-1β, were larger than 1.5, similar tothat of mono-specific bivalent binding. Upon the injection of the secondantigen, while DVD-Ig was already occupied by the first antigen, thestoichiometry of both DVD-Igs to the second antigen (i.e. hIL-1α orhIL-1β) was between 1.0 and 1.3. Thus DVD-Ig is able to bind two IL-1αand two IL-β molecules. DVD-Ig was first captured via a goat anti-humanFc antibody on the Biacore sensor chip, and the first antigen wasinjected and a binding signal observed, followed by the injection of thesecond antigen.

TABLE 14 Stoichiometry analysis of hIL-1a/b DVD-Ig in tetravalentdual-specific binding to IL-1α/β Stoichiometry Response Unit hIL-1α:hIL-1β: Captured Ab 1st antigen 2nd antigen DVD-Ig DVD-Ig DVD1-Ig: 932hIL-1α: 190 hIL-1β: 75 2.3 1.0 DVD1-Ig: 1092 hIL-1β: 141 hIL-1α: 107 1.11.5 DVD2-Ig: 1324 hIL-1α: 209 hIL-1β: 137 1.8 1.3 DVD2-Ig: 1184 hIL-1β:159 hIL-1α: 131 1.2 1.6

Example 1.7 Determination of Functional Homogeneity of DVD-Ig

Because DVD2-Ig was purified by Protein A chromatography instead oftarget-specific affinity chromatography, any potential misfolded and/ormismatched VL/VH domains, if present, can be assessed by binding studiesagainst the 2 different antigens. Such binding analysis was conduced bysize exclusion liquid chromatography (SEC). DVD2-Ig, alone or after a120-min incubation period at 37° C. with IL-1α, IL-1β, or both IL-1α andIL-1β, in equal molar ratio, were applied to the column. Each of theantigens was also run alone as controls. The SEC results indicated thatDVD2-Ig was able to bind IL-1α and IL-1β in solution, and such bindingresulted in a shift to the SEC signal indicating an increase in thedynamic size of DVD2-Ig when it was in complex with either antigen. Theshift of the DVD2-Ig signal was 100%, not partial, suggesting allDVD2-Ig molecules were able to bind the antigen. In the presence of bothIL-1α and IL-1β, there was a further and complete shift of the DVD2-Igsignal, indicating all DVD2-Ig molecules were able to bind both antigensin a uniform fashion. This experiment demonstrated that DVD-Ig wasexpressed as a functionally homogeneous protein. This has significantimplications as it demonstrates that DVD-Ig can be produced as ahomogeneous single, functional species, which differs from allpreviously described bi-specific, multi-specific, and multi-valentimmunoglobulin-like and immunoglobulin-derived molecules.

Example 1.8 Determination of Biological Activity of DVD-Ig

The biological activity of DVD-Ig was measured using MRC-5 bioassay. TheMRC-5 cell line is a human lung fibroblast cell line that produces IL-8in response to human IL-1α and IL-1β in a dose-dependent manner. MRC-5cells were obtained from ATCC and cultured in 10% FBS complete MEM at37° C. in a 5% CO2 incubator. To determine neutralizing activity of theDVD-Ig against human IL-1α or IL-1β, 50 ul of Ab (1E-7 to 1E-12 M) inMEM/10% FBS was added to a 96 well plate and pre-incubated with 50 ul ofhIL-1α or hIL-1β (200 pg/ml) for 1 hr at 37° C., 5% CO2. MRC-5 cells ata concentration of 1E5/ml were then added (100 ul) to all wells and theplates were incubated overnight at 37° C. in a 5% CO2 incubator. Thesupernatants were harvested, and human IL-8 production measured bystandard ELISA (R&D Systems, Minneapolis, Minn.). Neutralizing activityof the DVD-Ig was determined by its ability to inhibit IL-8 production.

As shown in Table 13, both DVD-Igs were able to neutralize hIL-1α andhIL-1β. Consistent with the binding affinity to hIL-1α, the neutralizingactivities of DVD1-Ig and DVD2-Ig against hIL-1α were also similar, i.e.3-fold less than that of the chimeric Abs (see Table III). Consistentwith its binding affinity for hIL-1β, the neutralizing activity ofDVD2-Ig to hIL-1β is slightly less than that of the chimeric Ab 13F5.G5,but 3-fold higher than that of DVD-Ig. Overall there was no significantdecrease in the biological activities of DVD-Ig molecules compared tothe original mAbs.

To determine if DVD-Ig was able to inhibit IL-8 production in thepresence of both IL-1α and IL-1β, equal amounts of hIL-1α and hIL-1βwere added in the same culture system of MRC-5 assay. Both DVD1-Ig andDVD2-Ig were able to inhibit IL-8 synthesis by MRC-5 cells in thepresence of both IL-1α and IL-1β, with activities similar to that ofmono-assays where only one cytokine was present (Table 13). In thisassay where both IL-1α and IL-1β were present, the dual-inhibitionactivity of DVD2-Ig (1.2 nM) was higher than that of DVD1-Ig (2.2 nM).

Example 2 Analysis of Linker Size and Variable Domain Orientation in theDVD-Ig Molecule

Additional DVD-Ig molecules with different parent mAb pairs, as shown inTable 15, were constructed. For each pair of mAbs, four different DVD-Igconstructs were generated: 2 with a short linker and 2 with a longlinker, each in two different domain orientations: a-b-C(alpha-beta-constant domain) and b-a-C (beta-alpha-constant domain). Thelinker sequences, were derived from the N-terminal sequence of human Ckor CH1 domain, as follows:

Short linker: light chain: TVAAP; (SEQ ID NO: 44) heavy chain: ASTKGP(SEQ ID NO: 42) Long linker: light chain: (SEQ ID NO: 50) TVAAPSVFIFPP;heavy chain: ASTKGPSVFPLAP (SEQ ID NO: 48).

All heavy and light chain constructs were subcloned into the pBOSexpression vector, and expressed in COS cells or freestyle 293 cells.

To construct new DVD clones, the variable domains of the two mAbs, bothlight chain and heavy chain, were first jointed in tandem usingoverlapping PCR as described for hIL-1abDVD1-Ig and hIL-1abDVD2-Ig. Thejointed pieces were then subcloned in pBOS vecter using homologousrecombination. Briefly, vectors were linearized by restriction digestion(2 ug of pBOS-hCk vector were digested with FspAI and BsiWI in O+buffer, and 2 ug of pBOS-hCγ z, non a vector was digested with FspAI andSail in O+ buffer). The digested samples were run on 1% agarose gel andthe backbone fragment purified in 50 ul water. For homologousrecombination and transformation, DH5α competent cells were thaw on ice,and mixed with 20-50 ng jointed PCR product and 20-50 ng of linearizedvector (in every 50 ul DH5α cells). The mixture was mixed gently andincubated on ice for 45 minutes, followed by heat shock at 42° C. for 1minute. Then 100 ul SOC medium were added and incubated at 37° C. for 1hour. The transformation culture was inoculated on LB/Agar platescontaining Ampicilin and incubated at 37° C. for 18-20 hours. Thebacterial clones were isolated, from which DNA was purified andsubjected to sequencing analysis. The final sequence-verified cloneswere co-transfected (matching HV and LC of the same Ab pair) in COS or293 cells for Ab expression and purification, as previously described.

Characteristics of the purified DVD-Ig proteins are summarized in Table16. The left section of the table 16 shows the specificity, bindingaffinity, and neutralization potency of the 2 pairs of mAbs used for theconstruction of the new hIL-1a/bDVD-Ig molecules. Antibodies 18F4.2C8and 1B12.4H4 (see example 1.1.D) were used to constructhIL-1a/bDVD3a-Ig, hIL-1a/bDVD4a-Ig, hIL-1a/bDVD3b-Ig, andhIL-1a/bDVD4b-Ig. hIL-1a/bDVD3a-Ig and hIL-1a/bDVD4a-Ig were in a-b-Corientation, with a short and long linker, respectively.hIL-1a/bDVD3b-Ig and hIL-1a/bDVD4b-Ig were in b-a-C orientation, with ashort and long linker, respectively. Antibodies 6H3.1A4 and 6B12.4F6were used to construct hIL-1a/bDVD5a-Ig, hIL-1a/bDVD6a-Ig,hIL-1a/bDVD5b-Ig, and hIL-1a/bDVD6b-Ig. hIL-1a/bDVD5a-Ig andhIL-1a/bDVD6a-Ig were in a-b-C orientation, with a short and longlinker, respectively. hIL-1a/bDVD5b-Ig and hIL-1a/bDVD6b-Ig were inb-a-C orientation, with a short and long linker, respectively. Themolecular cloning of these additional hIL-1a/bDVD-Igs were performedusing the procedure previously described for hIL-1a/bDVD1-Ig (seeexample 1.3), using overlapping PCR procedures. The amino acid sequencesof these additional hIL-1a/bDVD-Igs are disclosed in Table 15.

TABLE 15 Amino acid sequence of heavy chain and lightchain of six DVD Ig capable of binding IL-1α and IL-1β. Protein SequenceSequence Protein region Identifier 12345678901234567890 DVD HEAVYSEQ ID NO.: 41 EVQLQQSGAELVKPGASVKL VARIABLE SCTASGLNIKDTYMHWLKQRhIL-1a/b PEQGLEWIGRIDPANGNAKY DVD3a-Ig DPRFLGKATITADTSSNTAYLQLSSLTSEDTAVYYCARGD GNFHFDYWGQGTTLTVSSAS TKGPQVHLKESGPGLVAPSQSLSITCTVSGFSLTDYGVSW IRQPPGKGLEWLGLIWGGGD TYYNSPLKSRLSIRKDNSKSQVFLKMNSLQTDDTAVYYCA KQRTLWGYDLYGMDYWGQGT SVTVSS 18F4.2C8 VHSEQ ID NO.: 3 EVQLQQSGAELVKPGASVKL SCTASGLNIKDTYMHWLKQRPEQGLEWIGRIDPANGNAKY DPRFLGKATITADTSSNTAY LQLSSLTSEDTAVYYCARGDGNFHFDYWGQ GTTLTVSS LINKER SEQ ID NO.: 42 ASTKGP 1B12.4H4 VHSEQ ID NO.: 9 QVHLKESGPGLVAPSQSLSI TCTVSGFSLTDYGVSWIRQPPGKGLEWLGLIWGGGDTYYN SPLKSRLSIRKDNSKSQVFL KMNSLQTDDTAVYYCAKQRTLWGYDLYGMDYWGQGTSVTV SS CH SEQ ID NO.: 34 ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVS WNSGALTSGVHTFPAVLQSS GLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEP KSCDKTHTCPPCPAPELLGG PSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNW YVDGVEVHNAKTKPREEQYN STYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTIS KAKGQPREPQVYTLPPSREE MTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPV LDSDGSFFLYSKLTVDKSRW QQGNVFSCSVMHEALHNHYTQKSLSLSPGK DVD LIGHT SEQ ID NO.: 43 DIVMTQSQRFMSTSVGDRVS VARIABLE HIL-VTCKASQNVGTNIAWYQQKP 1a/b DVD3a-Ig GQSPRALIYSASYRYSGVPDRFTGSGSGTDFTLTISNVQS VDLAEYFCQQYTRYPLTFGG GTKLEIKRTVAAPETTVTQSPASLSMAIGEKVTIRCITST DIDVDMNWYQQKPGEPPKLL ISQGNTLRPGVPSRFSSSGSGTDFVFIIENMLSEDVADYY CLQSDNLPLTFGAGTKLELK RR 18F4.2C8 VL SEQ ID NO.: 4DIVMTQSQRFMSTSVGDRVS VTCKASQNVGTNIAWYQQKP GQSPRALIYSASYRYSGVPDRFTGSGSGTDFTLTISNVQS VDLAEYFCQQYTRYPLTFGG GTKLEIKR LINKER SEQ ID NO.: 44TVAAP 1B12.4H4 VL SEQ ID NO.: 10 ETTVTQSPASLSMAIGEKVTIRCITSTDIDVDMNWYQQKP GEPPKLLISQGNTLRPGVPS RFSSSGSGTDFVFIIENMLSEDVADYYCLQSDNLPLTFGA GTKLELKR CL SEQ ID NO.: 36 TVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQW KVDNALQSGNSQESVTEQDS KDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKS FNRGEC DVD HEAVY SEQ ID NO.: 45QVHLKESGPGLVAPSQSLSI VARIABLE TCTVSGFSLTDYGVSWIRQP hIL-1a/bPGKGLEWLGLIWGGGDTYYN DVD3b-Ig SPLKSRLSIRKDNSKSQVFL KMNSLQTDDTAVYYCAKQRTLWGYDLYGMDYWGQGTSVTV SSASTKGPEVQLQQSGAELV KPGASVKLSCTASGLNIKDTYMHWLKQRPEQGLEWIGRID PANGNAKYDPRFLGKATITA DTSSNTAYLQLSSLTSEDTAVYYCARGDGNFHFDYWGQGT TLTVSS 1B12.4H4 VH SEQ ID NO.: 9QVHLKESGPGLVAPSQSLSI TCTVSGFSLTDYGVSWIRQP PGKGLEWLGLIWGGGDTYYNSPLKSRLSIRKDNSKSQVFL KMNSLQTDDTAVYYCAKQRT LWGYDLYGMDYWGQGTSVTV SS LINKERSEQ ID NO.: 42 ASTKGP 18F4.2C8 VH SEQ ID NO.: 3 EVQLQQSGAELVKPGASVKLSCTASGLNIKDTYMHWLKQR PEQGLEWIGRIDPANGNAKY DPRFLGKATITADTSSNTAYLQLSSLTSEDTAVYYCARGD GNFHFDYWGQGTTLTVSS CH SEQ ID NO.: 34ASTKGPSVFPLAPSSKSTSG GTAALGCLVKDYFPEPVTVS WNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQT YICNVNHKPSNTKVDKKVEP KSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTP EVTCVVVDVSHEDPEVKFNW YVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGK EYKCKVSNKALPAPIEKTIS KAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDI AVEWESNGQPENNYKTTPPV LDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYT QKSLSLSPGK DVD LIGHT SEQ ID NO.: 46ETTVTQSPASLSMAIGEKVT VARIABLE HIL- IRCITSTDIDVDMNWYQQKP 1a/b DVD3b-IgGEPPKLLISQGNTLRPGVPS RFSSSGSGTDFVFIIENMLS EDVADYYCLQSDNLPLTFGAGTKLELKRTVAAPDIVMTQS QRFMSTSVGDRVSVTCKASQ NVGTNIAWYQQKPGQSPRALIYSASYRYSGVPDRFTGSGS GTDFTLTISNVQSVDLAEYF CQQYTRYPLTFGGGTKLEIKR1B12.4H4 VL SEQ ID NO.: 10 ETTVTQSPASLSMAIGEKVT IRCITSTDIDVDMNWYQQKPGEPPKLLISQGNTLRPGVPS RFSSSGSGTDFVFIIENMLS EDVADYYCLQSDNLPLTFGA GTKLELKRLINKER SEQ ID NO.: 44 TVAAP 18F4.2C8 VL SEQ ID NO.: 4DIVMTQSQRFMSTSVGDRVS VTCKASQNVGTNIAWYQQKP GQSPRALIYSASYRYSGVPDRFTGSGSGTDFTLTISNVQS VDLAEYFCQQYTRYPLTFGG GTKLEIKR CL SEQ ID NO.: 36TVAAPSVFIFPPSDEQLKSG TASVVCLLNNFYPREAKVQW KVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEK HKVYACEVTHQGLSSPVTKS FNRGEC DVD HEAVYSEQ ID NO.: 47 EVQLQQSGAELVKPGASVKL VARIABLE hIL- SCTASGLNIKDTYMHWLKQR1a/b DVD4a-Ig PEQGLEWIGRIDPANGNAKY DPRFLGKATITADTSSNTAYLQLSSLTSEDTAVYYCARGD GNFHFDYWGQGTTLTVSSAS TKGPSVFPLAPQVHLKESGPGLVAPSQSLSITCTVSGFSL TDYGVSWIRQPPGKGLEWLG LIWGGGDTYYNSPLKSRLSIRKDNSKSQVFLKMNSLQTDD TAVYYCAKQRTLWGYDLYGM DYWGQGTSVTVSS 18F4.2C8 VHSEQ ID NO.: 3 EVQLQQSGAELVKPGASVKL SCTASGLNIKDTYMHWLKQRPEQGLEWIGRIDPANGNAKY DPRFLGKATITADTSSNTAY LQLSSLTSEDTAVYYCARGDGNFHFDYWGQGTTLTVSS LINKER SEQ ID NO.: 48 ASTKGPSVFPLAP 1B12.4H4 VHSEQ ID NO.: 9 QVHLKESGPGLVAPSQSLSI TCTVSGFSLTDYGVSWIRQPPGKGLEWLGLIWGGGDTYYN SPLKSRLSIRKDNSKSQVFL KMNSLQTDDTAVYYCAKQRTLWGYDLYGMDYWGQGTSVTV SS CH SEQ ID NO.: 34 ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVS WNSGALTSGVHTFPAVLQSS GLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEP KSCDKTHTCPPCPAPELLGG PSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNW YVDGVEVHNAKTKPREEQYN STYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTIS KAKGQPREPQVYTLPPSREE MTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPV LDSDGSFFLYSKLTVDKSRW QQGNVFSCSVMHEALHNHYTQKSLSLSPGK DVD LIGHT SEQ ID NO.: 49 DIVMTQSQRFMSTSVGDRVS VARIABLE HIL-VTCKASQNVGTNIAWYQQKP 1a/bDVD4a-Ig GQSPRALIYSASYRYSGVPDRFTGSGSGTDFTLTISNVQS VDLAEYFCQQYTRYPLTFGG GTKLEIKRTVAAPSVFIFPPETTVTQSPASLSMAIGEKVT IRCITSTDIDVDMNWYQQKP GEPPKLLISQGNTLRPGVPSRFSSSGSGTDFVFIIENMLS EDVADYYCLQSDNLPLTFGA GTKLELKR 18F4.2C8 VLSEQ ID NO.: 4 DIVMTQSQRFMSTSVGDRVS VTCKASQNVGTNIAWYQQKPGQSPRALIYSASYRYSGVPD RFTGSGSGTDFTLTISNVQS VDLAEYFCQQYTRYPLTFGG GTKLEIKRLINKER SEQ ID NO.: 50 TVAAPSVFIFPP 1B12.4H4 VL SEQ ID NO.: 10ETTVTQSPASLSMAIGEKVT IRCITSTDIDVDMNWYQQKP GEPPKLLISQGNTLRPGVPSRFSSSGSGTDFVFIIENMLS EDVADYYCLQSDNLPLTFGA GTKLELKR CL SEQ ID NO.: 36TVAAPSVFIFPPSDEQLKSG TASVVCLLNNFYPREAKVQW KVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEK HKVYACEVTHQGLSSPVTKS FNRGEC DVD HEAVYSEQ ID NO.: 51 QVHLKESGPGLVAPSQSLSI VARIABLE hIL- TCTVSGFSLTDYGVSWIRQP1a/b DVD4b-Ig PGKGLEWLGLIWGGGDTYYN SPLKSRLSIRKDNSKSQVFLKMNSLQTDDTAVYYCAKQRT LWGYDLYGMDYWGQGTSVTV SSASTKGPSVFPLAPEVQLQQSGAELVKPGASVKLSCTAS GLNIKDTYMHWLKQRPEQGL EWIGRIDPANGNAKYDPRFLGKATITADTSSNTAYLQLSS LTSEDTAVYYCARGDGNFHF DYWGQGTTLTVSS 1B12.4H4 VHSEQ ID NO.: 9 QVHLKESGPGLVAPSQSLSI TCTVSGFSLTDYGVSWIRQPPGKGLEWLGLIWGGGDTYYN SPLKSRLSIRKDNSKSQVFL KMNSLQTDDTAVYYCAKQRTLWGYDLYGMDYWGQGTSVTV SS LINKER SEQ ID NO.: 48 ASTKGPSVFPLAP 18F4.2C8 VHSEQ ID NO.: 3 EVQLQQSGAELVKPGASVKL SCTASGLNIKDTYMHWLKQRPEQGLEWIGRIDPANGNAKY DPRFLGKATITADTSSNTAY LQLSSLTSEDTAVYYCARGDGNFHFDYWGQGTTLTVSS CH SEQ ID NO.: 34 ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVS WNSGALTSGVHTFPAVLQSS GLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEP KSCDKTHTCPPCPAPELLGG PSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNW YVDGVEVHNAKTKPREEQYN STYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTIS KAKGQPREPQVYTLPPSREE MTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPV LDSDGSFFLYSKLTVDKSRW QQGNVFSCSVMHEALHNHYTQKSLSLSPGK DVD LIGHT SEQ ID NO.: 52 ETTVTQSPASLSMAIGEKVT VARIABLE HIL-IRCITSTDIDVDMNWYQQKP 1a/b DVD4b-Ig GEPPKLLISQGNTLRPGVPSRFSSSGSGTDFVFIIENMLS EDVADYYCLQSDNLPLTFGA GTKLELKRTVAAPSVFIFPPDIVMTQSQRFMSTSVGDRVS VTCKASQNVGTNIAWYQQKP GQSPRALIYSASYRYSGVPDRFTGSGSGTDFTLTISNVQS VDLAEYFCQQYTRYPLTFGG GTKLEIKR 1B12.4H4 VLSEQ ID NO.: 10 ETTVTQSPASLSMAIGEKVT IRCITSTDIDVDMNWYQQKPGEPPKLLISQGNTLRPGVPS RFSSSGSGTDFVFIIENMLS EDVADYYCLQSDNLPLTFGA GTKLELKRLINKER SEQ ID NO.: 50 TVAAPSVFIFPP 18F4.2C8 VL SEQ ID NO.: 4DIVMTQSQRFMSTSVGDRVS VTCKASQNVGTNIAWYQQKP GQSPRALIYSASYRYSGVPDRFTGSGSGTDFTLTISNVQS VDLAEYFCQQYTRYPLTFGG GTKLEIKR CL SEQ ID NO.: 36TVAAPSVFIFPPSDEQLKSG TASVVCLLNNFYPREAKVQW KVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEK HKVYACEVTHQGLSSPVTKS FNRGEC DVD HEAVYSEQ ID NO.: 53 QVQLQQPGAELVRPGASVKL VARIABLE hIL- SCKASGYTFTTYWMNWVKQR1a/b DYD5a-Ig PEQGLEWIGRIDPYDSETLY SQKFKDTAILTVDKSSSTAYMQLSSLTSEDSAVYYCARYG FDYWGQGTTLTVSSASTKGP EVQLQQSGPELVKTGTSVKISCKASGYSFTGYYMHWVRQS HGKSLEWIGYISCYNGFTSY NPKFKGKATFTVDTSSSTAYIQFSRLTSEDSAVYYCARSD YYGTNDYWGQGTTLTVSS 6H3.1A4.3E11 SEQ ID NO.: 5QVQLQQPGAELVRPGASVKL VH SCKASGYTFTTYWMNWVKQR PEQGLEWIGRIDPYDSETLYSQKFKDTAILTVDKSSSTAY MQLSSLTSEDSAVYYCARYG FDYWGQGTTLTVSS LINKERSEQ ID NO.: 42 ASTKGP 6B12.4F6 VH SEQ ID NO.: 11 EVQLQQSGPELVKTGTSVKISCKASGYSFTGYYMHWVRQS HGKSLEWIGYISCYNGFTSY NPKFKGKATFTVDTSSSTAYIQFSRLTSEDSAVYYCARSD YYGTNDYWGQGTTLTVSS CH SEQ ID NO.: 34ASTKGPSVFPLAPSSKSTSG GTAALGCLVKDYFPEPVTVS WNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQT YICNVNHKPSNTKVDKKVEP KSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTP EVTCVVVDVSHEDPEVKFNW YVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGK EYKCKVSNKALPAPIEKTIS KAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDI AVEWESNGQPENNYKTTPPV LDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYT QKSLSLSPGK DVD LIGHT SEQ ID NO.: 54QIVLTQSPALMSASPGEKVT VARIABLE HIL- MTCSASSSVNYMYWYQQKPR 1a/b DVD5a-IgSSPKPWIYLTSNLASGVPAR FSGSGSGTSYSLTISSMEAE DAATYYCQQWNSNPYTFGGGTKLEMKRTVAAPQIVLTQSP AIMSASPGEKVTITCSASSS VSYMHWFQQKPGASPKLWIYSTSNLASGVPARFSGSGSGT SYSLTVSRMEAEDAATYYCQ QRSTYPYTFGGGTKLEIKR6H3.1A4.3E11 SEQ ID NO.: 6 QIVLTQSPALMSASPGEKVT VL MTCSASSSVNYMYWYQQKPRSSPKPWIYLTSNLASGVPAR FSGSGSGTSYSLTISSMEAE DAATYYCQQWNSNPYTFGGG TKLEMKRLINKER SEQ ID NO.: 44 TVAAP 6B12.4F6 VL SEQ ID NO.: 12QIVLTQSPAIMSASPGEKVT ITCSASSSVSYMHWFQQKPG ASPKLWIYSTSNLASGVPARFSGSGSGTSYSLTVSRMEAE DAATYYCQQRSTYPYTFGGG TKLEIKR CL SEQ ID NO.: 36TVAAPSVFIFPPSDEQLKSG TASVVCLLNNFYPREAKVQW KVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEK HKVYACEVTHQGLSSPVTKS FNRGEC DVD HEAVYSEQ ID NO.: 55 EVQLQQSGPELVKTGTSVKI VARIABLE hIL- SCKASGYSFTGYYMHWVRQS1a/b DVD5b-Ig HGKSLEWIGYISCYNGFTSY NPKFKGKATFTVDTSSSTAYIQFSRLTSEDSAVYYCARSD YYGTNDYWGQGTTLTVSSAS TKGPQVQLQQPGAELVRPGASVKLSCKASGYTFTTYWMNW VKQRPEQGLEWIGRIDPYDS ETLYSQKFKDTAILTVDKSSSTAYMQLSSLTSEDSAVYYC ARYGFDYWGQGTTLTVSS 6B12.4F6 VH SEQ ID NO.: 11EVQLQQSGPELVKTGTSVKI SCKASGYSFTGYYMHWVRQS HGKSLEWIGYISCYNGFTSYNPKFKGKATFTVDTSSSTAY IQFSRLTSEDSAVYYCARSD YYGTNDYWGQGTTLTVSS LINKERSEQ ID NO.: 42 ASTKGP 6H3.1A4.3E11 SEQ ID NO.: 5 QVQLQQPGAELVRPGASVKL VHSCKASGYTFTTYWMNWVKQR PEQGLEWIGRIDPYDSETLY SQKFKDTAILTVDKSSSTAYMQLSSLTSEDSAVYYCARYG FDYWGQGTTLTVSS CH SEQ ID NO.: 34ASTKGPSVFPLAPSSKSTSG GTAALGCLVKDYFPEPVTVS WNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQT YICNVNHKPSNTKVDKKVEP KSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTP EVTCVVVDVSHEDPEVKFNW YVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGK EYKCKVSNKALPAPIEKTIS KAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDI AVEWESNGQPENNYKTTPPV LDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYT QKSLSLSPGK DVD LIGHT SEQ ID NO.: 56QIVLTQSPAIMSASPGEKVT VARIABLE HIL- ITCSASSSVSYMHWFQQKPG 1a/b DVD5b-IgASPKLWIYSTSNLASGVPAR FSGSGSGTSYSLTVSRMEAE DAATYYCQQRSTYPYTFGGGTKLEIKRTVAAPQIVLTQSP ALMSASPGEKVTMTCSASSS VNYMYWYQQKPRSSPKPWIYLTSNLASGVPARFSGSGSGT SYSLTISSMEAEDAATYYCQ QWNSNPYTFGGGTKLEMKR6B12.4F6 VL SEQ ID NO.: 12 QIVLTQSPAIMSASPGEKVT ITCSASSSVSYMHWFQQKPGASPKLWIYSTSNLASGVPAR FSGSGSGTSYSLTVSRMEAE DAATYYCQQRSTYPYTFGGG TKLEIKRLINKER SEQ ID NO.: 44 TVAAP 6H3.1A4.3E11 SEQ ID NO.: 6QIVLTQSPALMSASPGEKVT VL MTCSASSSVNYMYWYQQKPR SSPKPWIYLTSNLASGVPARFSGSGSGTSYSLTISSMEAE DAATYYCQQWNSNPYTFGGG TKLEMKR CL SEQ ID NO.: 36TVAAPSVFIFPPSDEQLKSG TASVVCLLNNFYPREAKVQW KVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEK HKVYACEVTHQGLSSPVTKS FNRGEC DVD HEAVYSEQ ID NO.: 57 QVQLQQPGAELVRPGASVKL VARIABLE hIL- SCKASGYTFTTYWMNWVKQR1a/b DVD6a-Ig PEQGLEWIGRIDPYDSETLY SQKFKDTAILTVDKSSSTAYMQLSSLTSEDSAVYYCARYG FDYWGQGTTLTVSSASTKGP SVFPLAPEVQLQQSGPELVKTGTSVKISCKASGYSFTGYY MHWVRQSHGKSLEWIGYISC YNGFTSYNPKFKGKATFTVDTSSSTAYIQFSRLTSEDSAV YYCARSDYYGTNDYWGQGTT LTVSS 6H3.1A4.3E11SEQ ID NO.: 5 QVQLQQPGAELVRPGASVKL VH SCKASGYTFTTYWMNWVKQRPEQGLEWIGRIDPYDSETLY SQKFKDTAILTVDKSSSTAY MQLSSLTSEDSAVYYCARYGFDYWGQGTTLTVSS LINKER SEQ ID NO.: 48 ASTKGPSVFPLAP 6B12.4F6 VHSEQ ID NO.: 11 EVQLQQSGPELVKTGTSVKI SCKASGYSFTGYYMHWVRQSHGKSLEWIGYISCYNGFTSY NPKFKGKATFTVDTSSSTAY IQFSRLTSEDSAVYYCARSDYYGTNDYWGQGTTLTVSS CH SEQ ID NO.: 34 ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVS WNSGALTSGVHTFPAVLQSS GLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEP KSCDKTHTCPPCPAPELLGG PSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNW YVDGVEVHNAKTKPREEQYN STYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTIS KAKGQPREPQVYTLPPSREE MTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPV LDSDGSFFLYSKLTVDKSRW QQGNVFSCSVMHEALHNHYTQKSLSLSPGK DVD LIGHT SEQ ID NO.: 58 QIVLTQSPALMSASPGEKVT VARIABLE HIL-MTCSASSSVNYMYWYQQKPR 1a/b DVD 6a- SSPKPWIYLTSNLASGVPAR IgFSGSGSGTSYSLTISSMEAE DAATYYCQQWNSNPYTFGGG TKLEMKRTVAAPSVFIFPPQIVLTQSPAIMSASPGEKVTI TCSASSSVSYMHWFQQKPGA SPKLWIYSTSNLASGVPARFSGSGSGTSYSLTVSRMEAED AATYYCQQRSTYPYTFGGGT KLEIKRR 6H3.1A4.3E11SEQ ID NO.: 6 QIVLTQSPALMSASPGEKVT VL MTCSASSSVNYMYWYQQKPRSSPKPWIYLTSNLASGVPAR FSGSGSGTSYSLTISSMEAE DAATYYCQQWNSNPYTFGGG TKLEMKRLINKER SEQ ID NO.: 50 TVAAPSVFIFPP 6B12.4F6 VL SEQ ID NO.: 12QIVLTQSPAIMSASPGEKVT ITCSASSSVSYMHWFQQKPG ASPKLWIYSTSNLASGVPARFSGSGSGTSYSLTVSRMEAE DAATYYCQQRSTYPYTFGGG TKLEIKR CL SEQ ID NO.: 36RTVAAPSVFIFPPSDEQLKS GTASVVCLLNNFYPREAKVQ WKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYE KHKVYACEVTHQGLSSPVTK SFNRGEC DVD HEAVYSEQ ID NO.: 59 EVQLQQSGPELVKTGTSVKI VARIABLE hIL- SCKASGYSFTGYYMHWVRQS1a/b DVD6b-Ig HGKSLEWIGYISCYNGFTSY NPKFKGKATFTVDTSSSTAYIQFSRLTSEDSAVYYCARSD YYGTNDYWGQGTTLTVSSAS TKGPSVFPLAPQVQLQQPGAELVRPGASVKLSCKASGYTF TTYWMNWVKQRPEQGLEWIG RIDPYDSETLYSQKFKDTAILTVDKSSSTAYMQLSSLTSE DSAVYYCARYGFDYWGQGTT LTVSS 6B12.4F6 VHSEQ ID NO.: 11 EVQLQQSGPELVKTGTSVKI SCKASGYSFTGYYMHWVRQSHGKSLEWIGYISCYNGFTSY NPKFKGKATFTVDTSSSTAY IQFSRLTSEDSAVYYCARSDYYGTNDYWGQGTTLTVSS LINKER SEQ ID NO.: 48 ASTKGPSVFPLAP 6H3.1A4.3E11SEQ ID NO.: 5 QVQLQQPGAELVRPGASVKL VH SCKASGYTFTTYWMNWVKQRPEQGLEWIGRIDPYDSETLY SQKFKDTAILTVDKSSSTAY MQLSSLTSEDSAVYYCARYGFDYWGQGTTLTVSS CH SEQ ID NO.: 34 ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVS WNSGALTSGVHTFPAVLQSS GLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEP KSCDKTHTCPPCPAPELLGG PSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNW YVDGVEVHNAKTKPREEQYN STYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTIS KAKGQPREPQVYTLPPSREE MTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPV LDSDGSFFLYSKLTVDKSRW QQGNVFSCSVMHEALHNHYTQKSLSLSPGK DVD LIGHT SEQ ID NO.: 60 QIVLTQSPAIMSASPGEKVT VARIABLE HIL-ITCSASSSVSYMHWFQQKPG 1a/b DVD6b-Ig ASPKLWIYSTSNLASGVPARFSGSGSGTSYSLTVSRMEAE DAATYYCQQRSTYPYTFGGG TKLEIKRTVAAPSVFIFPPQIVLTQSPALMSASPGEKVTM TCSASSSVNYMYWYQQKPRS SPKPWIYLTSNLASGVPARFSGSGSGTSYSLTISSMEAED AATYYCQQWNSNPYTFGGGT KLEMKRR 6B12.4F6 VLSEQ ID NO.: 12 QIVLTQSPAIMSASPGEKVT ITCSASSSVSYMHWFQQKPGASPKLWIYSTSNLASGVPAR FSGSGSGTSYSLTVSRMEAE DAATYYCQQRSTYPYTFGGG TKLEIKRLINKER SEQ ID NO.: 50 TVAAPSVFIFPP 6H3.1A4.3E11 SEQ ID NO.: 6QIVLTQSPALMSASPGEKVT VL MTCSASSSVNYMYWYQQKPR SSPKPWIYLTSNLASGVPARFSGSGSGTSYSLTISSMEAE DAATYYCQQWNSNPYTFGGG TKLEMKR CL SEQ ID NO.: 36TVAAPSVFIFPPSDEQLKSG TASVVCLLNNFYPREAKVQW KVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEK HKVYACEVTHQGLSSPVTKS FNRGEC

Characteristics of the new DVD constructs are summarized in Table 16.Affinity (Kd) and biological activity (IC50) were determined by Biacoreand MRC-5 bioassay, respectively. SDS-PAGE analysis of all new DVDproteins showed normal migration patterns in both reduced andnon-reduced conditions, similar to a regular antibody and DVD1/2-Ig.

TABLE 16 Characterization of new DVD-Ig molecules derived from new mAbpairs Affinity Potency K_(d) IC₅₀ (K_(d)) M (IC₅₀) M mAb Specif. (M) (M)DVD Orient. Linker IL-1α IL-1β IL-1α IL-1β 18F4.2C8 rhIL-1α 5.95E−103.30E−10 DVD3a a-b-C short 8.37E−10 6.37E−08 7.50E−10 NA 1B12.4H4rhIL-1β 2.61E−10 6.00E−10 DVD4a a-b-C long 7.01E−10 9.30E−10 3.50E−101.00E−08 DVD3b b-a-C short 1.24E−09 1.90E−10 7.00E−10 4.00E−10 DVD4bb-a-C long 5.60E−10 1.28E−10 3.50E−10 5.00E−10 6H3.1A4 rhIL-1α 3.54E−102.40E−10 DVD5a a-b-C short 5.08E−10 1.25E−08 2.60E−09 1.90E−08 6B12.4F6rhIL-1β 5.54E−10 4.00E−10 DVD6a a-b-C long 1.06E−09 2.09E−09 2.30E−097.00E−08 DVD5b b-a-C short 1.32E−08 6.71E−10 3.30E−09 2.50E−10 DVD6bb-a-C long 8.20E−10 6.97E−10 1.00E−09 7.50E−10 NA: no neutralizationactivity detected.

The functional characterization of the new DVD molecules revealed thatwith either orientation, DVDs with the long linker performed better thanthe ones with the short linker in terms of binding and neutralizing ofboth antigens. With respect to DVDs with the long linkers, those withthe b-a-C orientation showed good binding to and neutralization of bothantigens, while the DVDs with an a-b-C orientation showed good bindingto and neutralization of IL-1α and reduced binding to and neutralizationof IL-1β (e.g. DVD4b vs. DVD4a). The DVD-Ig molecule, DVD4b, bound andneutralized both IL-1α and IL-1β with sub-nM and fully retained thebinding and neutralizing characteristics of the parent mAbs.

Example 3 Generation of DVD-Ig Capable of Binding IL-12 and IL-18

DVD-Ig molecules capable of binding IL-12 and IL-18 were constructed asdescribed above using two parent mAbs, one against human IL-12p40(ABT874), and the other against human IL-18 (ABT325). Four differentanti-IL12/18 DVD-Ig constructs were generated: 2 with short linker and 2with long linker, each in two different domain orientations: 12-18-C and18-12-C (Table VI). The linker sequences, derived from the N-terminalsequence of human C_(λ)/C_(κ) or CH1 domain, were as follows:

For DVD1218 constructs (ABT874 has a V_(λ)): (SEQ ID NO: 88) light chain(λ): Short linker: QPKAAP; (SEQ ID NO: 92) Long linker: QPKAAPSVTLFPP(SEQ ID NO: 42) heavy chain (γ1): Short linker: ASTKGP; (SEQ ID NO: 48)Long linker: ASTKGPSVFPLAP For DVD1812 constructs (ABT325 has a V_(κ)):(SEQ ID NO: 44) light chain (κ): Short linker: TVAAP; (SEQ ID NO: 50)Long linker: TVAAPSVFIFPP (SEQ ID NO: 42) heavy chain (γ1): Shortlinker: ASTKGP; (SEQ ID NO: 48) Long linker: ASTKGPSVFPLAP.

All heavy and light chain constructs were subcloned into the pBOSexpression vector, and expressed in COS cells or freestyle 293 cells,followed by purification by Protein A chromatography. The purifiedmaterials were subjected to SDS-PAGE and SEC, and their profiles weresimilar to that of the DVD2-Ig.

The table 17 below describes the heavy chain and light chain constructsused to express each anti-IL12/IL18 DVD-Ig protein.

TABLE 17 Constructs to express anti-IL12/IL18 DVD-Ig proteins DVD-Igprotein Heavy chain construct Light chain construct DVD1218SLDVD1218HC-SL DVD1218LC-SL DVD1218LL DVD1218HC-LL DVD1218LC-LL DVD1812SLDVD1812HC-SL DVD1812LC-SL DVD1812LL DVD1812HC-LL DVD1812LC-LL

Example 3.1.1 Molecular Cloning of DNA Constructs for DVD1218SL andDVD1218LL

To generate heavy chain constructs DVD1218HC-LL and DVD1218HC-SL, VHdomain of ABT-874 was PCR amplified using primers Primer 1 and Primer 2Lor Primer 2S respectively; meanwhile VH domain of ABT-325 was amplifiedusing primers Primer 3L or Primer 3S and Primer 4 respectively. Both PCRreactions were performed according to standard PCR techniques andprocedures. The two PCR products were gel-purified, and used together asoverlapping template for the subsequent overlapping PCR reaction usingprimers Primer 1 and Primer 4 using standard PCR conditions. Theoverlapping PCR products were subcloned into Srf I and Sal I doubledigested pBOS-hCγ1,z non-a mammalian expression vector (Abbott) by usingstandard homologous recombination approach.

To generate light chain constructs DVD1218LC-LL and DVD1218LC-SL, VLdomain of ABT-874 was PCR amplified using primers Primer 5 and Primer 6Lor Primer 6S respectively; meanwhile VL domain of ABT-325 was amplifiedusing primers Primer 7L or Primer 7S and Primer 8 respectively. Both PCRreactions were performed according to standard PCR techniques andprocedures. The two PCR products were gel-purified, and used together asoverlapping template for the subsequent overlapping PCR reaction usingprimers Primer 5 and Primer 8 using standard PCR conditions. Theoverlapping PCR products were subcloned into Srf I and Not I doubledigested pBOS-hCk mammalian expression vector (Abbott) by using standardhomologous recombination approach. The primers used for theseconstructions are listed below in table 18:

TABLE 18 Primer1: TAGAGATCCCTCGACCTCGAGATCCATTGTGCCCGGGCGCCA SEO ID NO.:61 CCATGGAGTTTGGGCTGAGC Primer2- SEQ ID NO.: 62 S:CACCTCTGGGCCCTTGGTCGACGCTGAAGAGACGGTGACCATTGT Primer2- SEQ ID NO.: 63 L:GGGTGCCAGGGGGAAGACCGATGGGCCCTTGGTCGACGCTGAAGA GACGGTGACCATTGT Primer3-SEQ ID NO.: 64 S: TCTTCAGCGTCGACCAAGGGCCCAGAGGTGCAGCTGGTGCAGTCT Primer3-SEQ ID NO.: 65 L: GCGTCGACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCGAGGTGCAGCTGGTGCAGTCT Primer 4: GTAGTCCTTGACCAGGCAGCC SEQ ID NO.: 66 Primer5:TAGAGATCCCTCGACCTCGAGATCCATTGTGCCCGGGCGCCA SEQ ID NO.: 67CCATGACTTGGACCCCACTC Primer 6- SEQ ID NO.: 68 S:TATTTCGGGGGCAGCCTTGGGCTGACCTAGTACTGTGACCTTGGT Primer6- SEQ ID NO.: 69 L:GGGCGGGAACAGAGTGACCGAGGGGGCAGCCTTGGGCTGACCTA GTACTGTGACCTTGGT Primer7-SEQ ID NO.: 70 S: CTAGGTCAGCCCAAGGCTGCCCCCGAAATAGTGATGACGCAGTCT Primer7-SEQ ID NO.: 71 L: CAGCCCAAGGCTGCCCCCTCGGTCACTCTGTTCCCGCCCGAAATAGTGATGACGCAGTCT Primer8: GTCCCAGGTGGGGACCCTCACTCTAGAGTCGCGGCCGCCTA SEQ IDNO.: 72 ACACTCTCCCCTGTTGAASimilar approach has been used to generate DVD 1812SL and DVD 1812LL asdescribed below:

Example 3.1.2 Molecular Cloning of DNA Constructs for DVD1812SL andDVD1812LL

To generate heavy chain constructs DVD 1812HC-LL and DVD 1812HC-SL, VHdomain of ABT-325 was PCR amplified using primers Primer 1 and Primer 9Lor Primer 9S respectively; meanwhile VH domain of ABT-874 was amplifiedusing primers Primer 10L or Primer 10S and Primer 4 respectively. BothPCR reactions were performed according to standard PCR techniques andprocedures. The two PCR products were gel-purified, and used together asoverlapping template for the subsequent overlapping PCR reaction usingprimers Primer 1 and Primer 4 using standard PCR conditions. Theoverlapping PCR products were subcloned into Srf I and Sal I doubledigested pBOS-hCγ1,z non-a mammalian expression vector (Abbott) by usingstandard homologous recombination approach. The following are primers'sequences:

To generate light chain constructs DVD1812LC-LL and DVD1812LC-SL, VLdomain of ABT-325 was PCR amplified using primers Primer 11 and Primer12L or Primer 12S respectively; meanwhile VL domain of ABT-874 wasamplified using primers Primer 13L or Primer 13S and Primer 14respectively. Both PCR reactions were performed according to standardPCR techniques and procedures. The two PCR products were gel-purified,and used together as overlapping template for the subsequent overlappingPCR reaction using primers Primer 11 and Primer 14 using standard PCRconditions. The overlapping PCR products were subcloned into Srf I andNot I double digested pBOS-hCk mammalian expression vector (Abbott) byusing standard homologous recombination approach. The primers used forthese constructions are listed below in table 19:

TABLE 19 Primer 9-S: SEQ ID NO.: 73CACCTGTGGGCCCTTGGTCGACGCTGAAGAGACGGTGACCATTGT Primer 9-L: SEQ ID NO.: 74GGGTGCCAGGGGGAAGACCGATGGGCCCTTGGTCGACGCTGAAGAG ACGGTGACCATTGT Primer 10-SEQ ID NO.: 75 S: TCTTCAGCGTCGACCAAGGGCCCACAGGTGCAGCTGGTGGAGTCT Primer10- SEQ ID NO.: 76 L: GCGTCGACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCCAGGTGCAGCTGGTGGAGTCT Primer 11: SEQ ID NO.: 77TAGAGATCCCTCGACCTCGAGATCCATTGTGCCCGGGCGCCACCATG GAAGCCCCAGCGCAGCTTPrimer 12-S: SEQ ID NO.: 78 AGACTGTGGTGCAGCCACAGTTCGTTTAATCTCCAGTCGTGTPrimer 12- SEQ ID NO.: 79 L:TGGCGGGAAGATGAAGAGAGATGGTGCAGCCACAGTTCGTTTAAT CTCCAGTCGTGT Primer 13-S:SEQ ID NO.: 80 AAACGAACTGTGGCTGCACCACAGTCTGTGCTGACTCAGCCC Primer 13- SEQID NO.: 81 L: ACTGTGGCTGCACCATCTGTCTTCATCTTCCCGCCACAGTCTGTGC TGACTCAGCCCPrimer 14: SEQ ID NO.: 82 GTCCCAGGTGGGGACCCTCACTCTAGAGTCGCGGCCGCTCATGAACATTCTGTAGGGGC

The final DNA sequences for eight heavy and light chain constructs ofanti-IL12/IL-18 DVD-Ig are as shown in table 20:

TABLE 20 Amino acid sequence of DVD binding proteins capable of bindingIL-12 and IL-18 Protein Sequence Sequence Protein region Identifier12345678901234567890 DVD HEAVY SEQ ID NO.: 83 QVQLVESGGGVVQPGRSLRLVARIABLE SCAASGFTFSSYGMHWVRQA DVD1218HC-SL PGKGLEWVAFIRYDGSNKYYADSVKGRFTISRDNSKNTLY LQMNSLRAEDTAVYYCKTHG SHDNWGQGTMVTVSSASTKGPEVQLVQSGTEVKKPGESLK ISCKGSGYTVTSYWIGWVRQ MPGKGLEWMGFIYPGDSETRYSPTFQGQVTISADKSFNTA FLQWSSLKASDTAMYYCARV GSGWYPYTFDIWGQGTMVTV SSABT-874 VH SEQ ID NO.: 84 QVQLVESGGGVVQPGRSLRL SCAASGFTFSSYGMHWVRQAPGKGLEWVAFIRYDGSNKYY ADSVKGRFTISRDNSKNTLY LQMNSLRAEDTAVYYCKTHGSHDNWGQGTMVTVSS LINKER SEQ ID NO.: 42 ASTKGP ABT-325 VH SEQ ID NO.: 85EVQLVQSGTEVKKPGESLKI SCKGSGYTVTSYWIGWVRQM PGKGLEWMGFIYPGDSETRYSPTFQGQVTISADKSFNTAF LQWSSLKASDTAMYYCARVG SGWYPYTFDIWGQGTMVTVSS CH SEQID NO.: 34 ASTKGPSVFPLAPSSKSTSG GTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSS GLYSLSSVVTVPSSSLGTQT YICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGG PSVFLFPPKPKDTLMISRTP EVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYN STYRVVSVLTVLHQDWLNGK EYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREE MTKNQVSLTCLVKGFYPSDI AVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRW QQGNVFSCSVMHEALHNHYT QKSLSLSPGK DVD LIGHT SEQ IDNO.: 86 MTWTPLLFLTLLLHCTGSLS VARIABLE QSVLTQPPSVSGAPGQRVTI DVD1218LC-SLSCSGSRSNIGSNTVKWYQQL PGTAPKLLIYYNDQRPSGVP DRFSGSKSGTSASLAITGLQAEDEADYYCQSYDRYTHPAL LFGTGTKVTVLGQPKAAPEI VMTQSPATLSVSPGERATLSCRASESISSNLAWYQQKPGQ APRLFIYTASTRATDIPARF SGSGSGTEFTLTISSLQSEDFAVYYCQQYNNWPSITFGQG TRLEIKR ABT-874 VL SEQ ID NO.: 87QSVLTQPPSVSGAPGQRVTI SCSGSRSNIGSNTVKWYQQL PGTAPKLLIYYNDQRPSGVPDRFSGSKSGTSASLAITGLQ AEDEADYYCQSYDRYTHPAL LFGTGTKVTVLG LINKER SEQ IDNO.: 88 QPKAAP ABT-325 VL SEQ ID NO.: 89 EIVMTQSPATLSVSPGERATLSCRASESISSNLAWYQQKP GQAPRLFIYTASTRATDIPA RFSGSGSGTEFTLTISSLQSEDFAVYYCQQYNNWPSITFG QGTRLEIKR CL SEQ ID NO.: 36 TVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQW KVDNALQSGNSQESVTEQDS KDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKS FNRGEC DVD HEAVY SEQ ID NO.: 90QVQLVESGGGVVQPGRSLRL SCAASGFTFSSYGMHWVRQA PGKGLEWVAFIRYDGSNKYYADSVKGRFTISRDNSKNTLY VARIABLE LQMNSLRAEDTAVYYCKTHG DVD1218HC-LLSHDNWGQGTMVTVSSASTKG PSVFPLAPEVQLVQSGTEVK KPGESLKISCKGSGYTVTSYWIGWVRQMPGKGLEWMGFIY PGDSETRYSPTFQGQVTISA DKSFNTAFLQWSSLKASDTAMYYCARVGSGWYPYTFDIWG QGTMVTVSS ABT-874 VH SEQ ID NO.: 84QVQLVESGGGVVQPGRSLRL SCAASGFTFSSYGMHWVRQA PGKGLEWVAFIRYDGSNKYYADSVKGRFTISRDNSKNTLY LQMNSLRAEDTAVYYCKTHG SHDNWGQGTMVTVSS LINKER SEQ IDNO.: 48 ASTKGPSVFPLAP ABT-325 VH SEQ ID NO.: 85 EVQLVQSGTEVKKPGESLKISCKGSGYTVTSYWIGWVRQM PGKGLEWMGFIYPGDSETRY SPTFQGQVTISADKSFNTAFLQWSSLKASDTAMYYCARVG SGWYPYTFDIWGQGTMVTVSS CH SEQ ID NO.: 34ASTKGPSVFPLAPSSKSTSG GTAALGCLVKDYFPEPVTVS WNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQT YICNVNHKPSNTKVDKKVEP KSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTP EVTCVVVDVSHEDPEVKFNW YVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGK EYKCKVSNKALPAPIEKTIS KAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDI AVEWESNGQPENNYKTTPPV LDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYT QKSLSLSPGK DVD LIGHT SEQ ID NO.: 91QSVLTQPPSVSGAPGQRVTI VARIABLE SCSGSRSNIGSNTVKWYQQL DVD1218LC-LLPGTAPKLLIYYNDQRPSGVP DRFSGSKSGTSASLAITGLQ AEDEADYYCQSYDRYTHPALLFGTGTKVTVLGQPKAAPSV TLFPPEIVMTQSPATLSVSP GERATLSCRASESISSNLAWYQQKPGQAPRLFIYTASTRA TDIPARFSGSGSGTEFTLTI SSLQSEDFAVYYCQQYNNWPSITFGQGTRLEIKR ABT-874 VL SEQ ID NO.: 87 QSVLTQPPSVSGAPGQRVTISCSGSRSNIGSNTVKWYQQL PGTAPKLLIYYNDQRPSGVP DRFSGSKSGTSASLAITGLQAEDEADYYCQSYDRYTHPAL LFGTGTKVTVLG LINKER SEQ ID NO.: 92 QPKAAPSVTLFPPABT-325 VL SEQ ID NO.: 89 EIVMTQSPATLSVSPGERAT LSCRASESISSNLAWYQQKPGQAPRLFIYTASTRATDIPA RFSGSGSGTEFTLTISSLQS EDFAVYYCQQYNNWPSITFG QGTRLEIKRCL SEQ ID NO.: 36 TVAAPSVFIFPPSDEQLKSG TASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDS KDSTYSLSSTLTLSKADYEK HKVYACEVTHQGLSSPVTKS FNRGECDVD HEAVY SEQ ID NO.: 93 EVQLVQSGTEVKKPGESLKI VARIABLESCKGSGYTVTSYWIGWVRQM DVD1812HC-SL PGKGLEWMGFIYPGDSETRYSPTFQGQVTISADKSFNTAF LQWSSLKASDTAMYYCARVG SGWYPYTFDIWGQGTMVTVSSASTKGPQVQLVESGGGVVQ PGRSLRLSCAASGFTFSSYG MHWVRQAPGKGLEWVAFIRYDGSNKYYADSVKGRFTISRD NSKNTLYLQMNSLRAEDTAV YYCKTHGSHDNWGQGTMVTV SSABT-325 VH SEQ ID NO.: 85 EVQLVQSGTEVKKPGESLKI SCKGSGYTVTSYWIGWVRQMPGKGLEWMGFIYPGDSETRY SPTFQGQVTISADKSFNTAF LQWSSLKASDTAMYYCARVGSGWYPYTFDIWGQGTMVTVSS LINKER SEQ ID NO.: 42 ASTKGP ABT-874 VH SEQ IDNO.: 84 QVQLVESGGGVVQPGRSLRL SCAASGFTFSSYGMHWVRQA PGKGLEWVAFIRYDGSNKYYADSVKGRFTISRDNSKNTLY LQMNSLRAEDTAVYYCKTHG SHDNWGQGTMVTVSS CH SEQ ID NO.:34 ASTKGPSVFPLAPSSKSTSG GTAALGCLVKDYFPEPVTVS WNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQT YICNVNHKPSNTKVDKKVEP KSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTP EVTCVVVDVSHEDPEVKFNW YVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGK EYKCKVSNKALPAPIEKTIS KAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDI AVEWESNGQPENNYKTTPPV LDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYT QKSLSLSPGK DVD LIGHT SEQ ID NO.: 94EIVMTQSPATLSVSPGERAT VARIABLE LSCRASESISSNLAWYQQKP DVD1812LC-SLGQAPRLFIYTASTRATDIPA RFSGSGSGTEFTLTISSLQS EDFAVYYCQQYNNWPSITFGQGTRLEIKRTVAAPQSVLTQ PPSVSGAPGQRVTISCSGSR SNIGSNTVKWYQQLPGTAPKLLIYYNDQRPSGVPDRFSGS KSGTSASLAITGLQAEDEAD YYCQSYDRYTHPALLFGTGT KVTVLGABT-325 VL SEQ ID NO.: 89 EIVMTQSPATLSVSPGERAT LSCRASESISSNLAWYQQKPGQAPRLFIYTASTRATDIPA RFSGSGSGTEFTLTISSLQS EDFAVYYCQQYNNWPSITFG QGTRLEIKRLINKER SEQ ID NO.: 44 TVAAP ABT-874 VL SEQ ID NO.: 87QSVLTQPPSVSGAPGQRVTI SCSGSRSNIGSNTVKWYQQL PGTAPKLLIYYNDQRPSGVPDRFSGSKSGTSASLAITGLQ AEDEADYYCQSYDRYTHPAL LFGTGTKVTVLG CL SEQ ID NO.: 36TVAAPSVFIFPPSDEQLKSG TASVVCLLNNFYPREAKVQW KVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEK HKVYACEVTHQGLSSPVTKS FNRGEC DYD HEAVY SEQ ID NO.:95 EVQLVQSGTEVKKPGESLKI VARIABLE SCKGSGYTVTSYWIGWVRQM DVD1812HC-LLPGKGLEWMGFIYPGDSETRY SPTFQGQVTISADKSFNTAF LQWSSLKASDTAMYYCARVGSGWYPYTFDIWGQGTMVTVS SASTKGPSVFPLAPQVQLVE SGGGVVQPGRSLRLSCAASGFTFSSYGMHWVRQAPGKGLE WVAFIRYDGSNKYYADSVKG RFTISRDNSKNTLYLQMNSLRAEDTAVYYCKTHGSHDNWG QGTMVTVSS ABT-325 VH SEQ ID NO.: 85EVQLVQSGTEVKKPGESLKI SCKGSGYTVTSYWIGWVRQM PGKGLEWMGFIYPGDSETRYSPTFQGQVTISADKSFNTAF LQWSSLKASDTAMYYCARVG SGWYPYTFDIWGQGTMVTVSS LINKERSEQ ID NO.: 48 ASTKGPSVFPLAP ABT-875 VH SEQ ID NO.: 84QVQLVESGGGVVQPGRSLRL SCAASGFTFSSYGMHWVRQA PGKGLEWVAFIRYDGSNKYYADSVKGRFTISRDNSKNTLY LQMNSLRAEDTAVYYCKTHG SHDNWGQGTMVTVSS CH SEQ ID NO.:34 ASTKGPSVFPLAPSSKSTSG GTAALGCLVKDYFPEPVTVS WNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQT YICNVNHKPSNTKVDKKVEP KSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTP EVTCVVVDVSHEDPEVKFNW YVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGK EYKCKVSNKALPAPIEKTIS KAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDI AVEWESNGQPENNYKTTPPV LDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYT QKSLSLSPGK DVD LIGHT SEQ ID NO.: 96EIVMTQSPATLSVSPGERAT VARIABLE LSCRASESISSNLAWYQQKP DVD1812LC-LLGQAPRLFIYTASTRATDIPA RFSGSGSGTEFTLTISSLQS EDFAVYYCQQYNNWPSITFGQGTRLEIKRTVAAPSVFIFP PQSVLTQPPSVSGAPGQRVT ISCSGSRSNIGSNTVKWYQQLPGTAPKLLIYYNDQRPSGV PDRFSGSKSGTSASLAITGL QAEDEADYYCQSYDRYTHPALLFGTGTKVTVLG ABT-325 VL SEQ ID NO.: 89 EIVMTQSPATLSVSPGERATLSCRASESISSNLAWYQQKP GQAPRLFIYTASTRATDIPA RFSGSGSGTEFTLTISSLQSEDFAVYYCQQYNNWPSITFG QGTRLEIKR LINKER SEQ ID NO.: 50 TVAAPSVFIFPPABT-874 VL SEQ ID NO.: 87 QSVLTQPPSVSGAPGQRVTI SCSGSRSNIGSNTVKWYQQLPGTAPKLLIYYNDQRPSGVP DRFSGSKSGTSASLAITGLQ AEDEADYYCQSYDRYTHPALLFGTGTKVTVLG CL SEQ ID NO.: 36 TVAAPSVFIFPPSDEQLKSG TASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDS KDSTYSLSSTLTLSKADYEK HKVYACEVTHQGLSSPVTKS FNRGEC

Example 3.2 Determination of Antigen Binding Affinity of IL-12/IL-18 DVDIgs

The binding affinity of anti-IL-12/18 DVD-Igs to hIL-12 and hIL-18 weredetermined by Biacore (Table 21). The neutralization activity againstIL-18 was determined by KG-1 assay (Konishi, K., et al.,). Briefly,IL-18 samples (in a final concentration of 2 ng/ml) were pre-incubatedwith DVD-Ig (in final concentrations between 0 and 10 mg/ml) at 37° C.for 1 hr, and then added to KG-1 cells (3×10⁶/ml) in RPMI mediumcontaining 10 ng/ml hTNF, followed by incubation at 37° C. for 16-20 hr.The culture supernatants were collected and human IFN-γ production ineach sample was determined by ELISA (R&D Systems). Inhibition activitiesof the DVD molecules against IL-18, presented as IC₅₀ values, are shownin Table VI. To determine the inhibition activities of anti-IL-12/18 DVDmolecules against IL-12, an IL-12-induced IFN-γ production assay fromactivated PHA blast cells was employed (D'Andrea, A et al.,) Forproduction of human IFN-γ, PHA blast cells were incubated for 18 hourswith human IL-12. Sub-maximal stimulation (55-75% of maximum) wasobtained with a human IL-12 concentration of 200 pg/mL. Supernatantswere assayed for IFN-γ using a specific human IFN-γ ELISA (Endogen,Cambridge, Mass.). Neutralizing IL-12 DVDs interfere with IL-12 inducedIFN-γ production. The neutralization activity of DVD is determined bymeasuring the DVD concentration required to inhibit 50% of the IFN-γproduction by human PHA blast cells, as shown in Table 21.

TABLE 21 Characterization of anti-IL-18/IL-12 DVD-Ig molecules AffinityPotency K_(d) IC₅₀ (K_(d), M) (IC₅₀, M) MAb Specif. (M) (M) DVD Orient.Linker IL-12 IL-18 IL-12 IL-18 ABT874 hIL- 6.47E−11 5.0E−12 DVD1218-12-18-C short 3.81E−11 6.22E−10 6.93E−12 1.8E−10 12 SL ABT325 hIL-1.37E−10 3.0E−10 DVD1218- 12-18-C long 2.38E−11 6.64E−10 3.04E−121.8E−10 18 LL DVD1812- 18-12-C short 1.82E−09 1.91E−10 3.66E−10 4.0E−11SL DVD1812- 18-12-C long 1.13E−10 1.62E−10 1.18E−10 7.8E−11 LL Affinity(Kd) was determined by Biacore and potency (IC50) determined by KG-1bioassay (IL-18) and PBMC assay (IL-12).

Table 21 shows the specificity, binding affinity, and neutralizationactivity of the 2 fully human mAbs used for the construction of theanti-IL-12/IL-18 DVD molecules. As shown in the Table VI, these mAbshave high affinity and neutralization activity. A summary of thecharacterization of the anti-IL-18/IL-12 DVD constructs is shown inTable VI. SDS-PAGE analysis of all new DVD proteins showed normalmigration patterns in both reduced and non-reduced conditions, similarto a regular antibody and DVD1/2-Ig. SEC analysis indicated allmolecules were normal, exhibiting peaks in the 200 kD region. TheBiacore binding data are consistent with the neutralization activity inthe biological assays.

Example 3.3 Biological Activity of anti-IL-12/IL-18 DVD-Ig In Vivo

Both IL-12 and IL-18 are required to produce optimal IFNγ in response tovarious stimuli. The biological activity of anti-IL-12/IL-18 DVD-Ig invivo was determined using the huPBMC-SCID mouse model. In this model,anti-IL-12 antibody (ABT-874) anti-IL-18 antibody (ABT-325) or theanti-IL-12/anti-IL-18 DVD-Ig were injected i.p. or i.v. (250 mg/mouseeach) followed by transfer of freshly purified human PBMCs (huPBMC) i.p.into SCID mice. Fifteen minutes later, mice were challenged with driedstaphylococcus aureus cells (SAC) to induce human IFNγ production.Animals (n=7-8/group) were sacrificed 18-20 hrs later and serum huIFNγlevels were determined by ELISA. ABT 874 and ABT-325 inhibitedSAC-induced IFNγ by approximately 70% which represents maximum IFNγinhibition with each compound in this model. However, treatment of micewith ABT-874+ABT-325 and anti-IL-12/anti-IL-18 DVD-Ig inhibited IFNγproduction by almost 100%. These results suggest that theanti-IL-12/anti-IL-18 DVD-Ig molecule is functionally active in vivo.

Example 3.4 Pharmacokinetic and Pharmacodynamic Studies ofAnti-IL-12/IL-18 DVD-Ig

The overall Pharmacokinetic and pharmacodynamic profile ofanti-IL-12/IL-18 DVD-Ig was similar to the parent mAbs in mice, i.e 73%bioavailability, comparable to regular IgG. Similar pharmacokinetics,i.e. rapid clearance after day 6-8, was also observed for other mAbs(e.g. human, rat etc,) probably due to anti-human IgG response.

Male SD rats were dosed with anti-IL-12/IL-18 DVD-Ig at 4 mg/kg eitheri.v. or s.c. The early part of the PK curves looked normal and verysimilar to those of other human antibodies. An accurate half-life inboth groups could not be derived because of the rapid clearance ofDVD-Ig beginning on day 6. The sudden drop in DVD-Ig concentration afterday 6 may be due to the RAHA response. However, similar profile has alsobeen observed for one of the parent antibodies (ABT-874) used forconstruction of this DVD-Ig in this particular experiment, as well asother mono-specific human antibodies previously studied. Based on DVD-Igconcentration up to day 6 in both s.c and i.v. groups, bioavailabilityof DVD-Ig was estimated. Two out of three rats showed 80-95%bioavailability, and the average bioavailability in the three mice was73%

Example 3.5 Physical/Chemical Characterization of Anti-IL-12/Anti-IL-18DVD-Ig

Results of physical and chemical characterization of 293 cell-derived,protein A purified, anti-IL-12/anti-IL-18 DVD-Ig are summarized in Table22.

TABLE 22 Physical/Chemical Characterization of anti-IL-12/anti-IL-18DVD-Ig Parameters Tested Assay/Methodology Findings/Comments Affinity(Kd) IL-12 Biacore 38 pM (65 pM for ABT-874) IL-18 Biacore 622 pM (137pM for ABT-325) Potency (IC50) IL-12 PHA-Blast Assay 7 pM (5 pM forABT874) IL-18 KG-1 Assay 180 pM (300 pM for ABT-325) M.W MS HC: 64130(theo. 64127) LC: 36072 (theo. 36072) Amino acid sequence Sequencing -MS All matched Disulfide bonds Peptide mapping All 20 disulfide bondsare matched Glycosylation profile Similar to other in-house fully humanantibodies - NGA2F and NGA1F observed as the major forms Charge CationExchange Homogeneity heterogeneity (WCX-10) PI cIEF 9.42 (ABT-874: 9.46)Dynamic size DSL 7.69 nM (5.34 nM for ABT-325) Purity/aggregates SDS-PGEHomogeneity on both reducing (~64 Kd HC and ~36 Kd LC bands) andnon-reducing (one SEC band) gels AUC One peak (~100%) observedimmediately after protein A purification by SEC ~16-17% aggregatesobserved after 2 cycles of freeze-thaw by AUC Stability SEC ~5%aggregates after 2 freeze-thaw cycles, (freeze/thaw) increased to ~13%after additional 10 freeze-thaw cycles. The reason for that is unsolved(process- related, sequence-specific, or LC lamda/kappa hybrid) PKprofile Rat i.v. & s.c. Similar to (or limited by) parental mAbs.Bioavailability Rat i.v. vs s.c. Average 73%; Overall similar toparental mAbs

Example 3.6 Generation of an Additional Anti-12/Anti-18 DVD-Ig(1D4.1-ABT325)

An additional anti-IL-12/IL-18 DVD-Ig molecule with a different parentanti-IL-12 mAb (clone#1D4.1), as shown in Table 23, was constructed. The1D4.1-ABT325 DVD-Ig construct was generated with a short linker derivedfrom the N-terminal sequence of human Ck and CH1 domain, as follows:

Short linker: light chain: TVAAP (SEQ ID NO: 44); heavy chain: ASTKGP(SEQ ID NO: 42).

All heavy and light chain constructs were subcloned into the pBOSexpression vector, expressed in COS cells or freestyle 293 cells, andcharacterized as described above. 1D4.1-ABT325 DVD-Ig fully retains theactivities of the two original mAbs (Table 24).

TABLE 23 Amino acid sequence of 1D4.1-ABT325 DVD-Ig Protein ProteinSequence Sequence region Identifier 12345678901234567890 1D4.1-ABT325SEQ ID NO.: 114 EVTLRESGPALVKPTQTLTL DVD-Ig HEAVY TCTFSGFSLSKSVMGVSWIRVARIABLE QPPGKALEWLAHIYWDDDKY YNPSLKSRLTISKDTSKNQV VLTMTNMDPVDTATYYCARRGIRSAMDYWGQGTTVTVSSA STKGPEVQLVQSGTEVKKPG ESLKISCKGSGYTVTSYWIGWVRQMPGKGLEWMGFIYPGD SETRYSPTFQGQVTISADKS FNTAFLQWSSLKASDTAMYYCARVGSGWYPYTFDIWGQGT MVTVSS 1D4.1 VH SEQ ID NO.: 115EVTLRESGPALVKPTQTLTL TCTFSGFSLSKSVMGVSWIR QPPGKALEWLAHIYWDDDKYYNPSLKSRLTISKDTSKNQV VLTMTNMDPVDTATYYCARR GIRSAMDYWGQGTTVTVSS LINKER SEQID NO.: 42 ASTKGP ABT-325 VH SEQ ID NO.: 85 EVQLVQSGTEVKKPGESLKISCKGSGYTVTSYWIGWVRQM PGKGLEWMGFIYPGDSETRY SPTFQGQVTISADKSFNTAFLQWSSLKASDTAMYYCARVG SGWYPYTFDIWGQGTMVTVSS CH SEQ ID NO.: 34ASTKGPSVFPLAPSSKSTSG GTAALGCLVKDYFPEPVTVS WNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQT YICNVNHKPSNTKVDKKVEP KSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTP EVTCVVVDVSHEDPEVKFNW YVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGK EYKCKVSNKALPAPIEKTIS KAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDI AVEWESNGQPENNYKTTPPV LDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYT QKSLSLSPGK 1D4.1-ABT325 SEQ ID NO.: 116DIVMTQSPDSLAVSLGERAT DVD-Ig LIGHT INCKASQSVSNDVAWYQQKP VARIABLEGQPPKLLIYYASNRYTGVPD RFSGSGSGTDFTLTISSLQA EDVAVYYCQQDYNSPWTFGGGTKVEIKRTVAAPEIVMTQS PATLSVSPGERATLSCRASE SISSNLAWYQQKPGQAPRLFIYTASTRATDIPARFSGSGS GTEFTLTISSLQSEDFAVYY CQQYNNWPSITFGQGTRLEI KR1D4.1VL SEQ ID NO.: 117 DIVMTQSPDSLAVSLGERAT INCKASQSVSNDVAWYQQKPGQPPKLLIYYASNRYTGVPD RFSGSGSGTDFTLTISSLQA EDVAVYYCQQDYNSPWTFGG GTKVEIKRLINKER SEQ ID NO.: 44 TVAAP ABT-325 VL SEQ ID NO.: 89EIVMTQSPATLSVSPGERAT LSCRASESISSNLAWYQQKP GQAPRLFIYTASTRATDIPARFSGSGSGTEFTLTISSLQS EDFAVYYCQQYNNWPSITFG QGTRLEIKR CL SEQ ID NO.: 36TVAAPSVFIFPPSDEQLKSG TASVVCLLNNFYPREAKVQW KVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEK HKVYACEVTHQGLSSPVTKS FNRGEC

TABLE 24 Characterization 1D4.1-ABT325 DVD-Ig molecule Affinity (K_(d),M) Potency (IC₅₀, M) mAb IL-12 IL-18 IL-12 IL-18 1D4.1 1.20E−10 N/A4.18E−10 N/A ABT325 N/A 1.91E−10 N/A 6.87E−11 1D4.1-ABT325 DVD-Ig1.33E−10 1.59E−10 2.17E−10 1.20E−10 Affinity (Kd) was determined byBiacore and potency (IC50) determined by KG-1 bioassay (IL-18) and PBMCassay (IL-12).

Example 4 Generation of Anti-CD20/Anti-CD3 DVD-Ig

Anti-CD20/anti-CD3 DVD-Igs were generated using murine anti-human-CD20(clone 5F1) and anti-human-CD3 (clone OKT3) parent antibodies. Theinitial constructs included 2 DVD-Igs with different domainorientations. The anti-CD3/anti-CD20 DVD-Ig was constructed in the orderof V_(CD3)-linker-VCD20-constant, and anti-CD20/anti-CD3 DVD-Ig wasconstructed in the order of V_(CD20)-linker-V_(CD3)-constant. However,in a preliminary cell surface binding study, anti-CD20 binding activitywas diminished in the anti-CD3/anti-CD20 DVD-Ig molecule, even thoughthe anti-CD3 activity was conserved in this design. In contrast, bothanti-CD3 and anti-CD20 binding activities were fully conserved in theanti-CD20/anti-CD3 DVD-Ig molecule, indicating this is the optimaldomain orientation for these two mAbs/targets combination in a DVD-Igformat. Therefore the anti-CD20/anti-CD3 DVD-Ig construct was chosen forsubsequent studies.

The anti-CD20/anti-CD3 DVD-Ig was generated as chimeric Ig i.e theconstant region was a human constant region. For binding analysis, humanT cell line Jurkat and B cell line Raji were blocked with human IgG andthen stained with murine anti-hCD3 mAb OKT3, murine anti-hCD20 mAb 1F5,and anti-CD20/anti-CD3 DVD-Ig. Cells were then washed and stained withFITC-labeled goat anti-murine IgG (with no anti-hIgG cross-reactivity).Anti-CD20/CD3 DVD-Ig bound both T and B cells, whereas CD3 and CD20 mAbsbound only T or B cells, respectively. The amino acid sequence ofCD20/CD3 DVD-Ig is disclosed in Table 25.

TABLE 25 Amino acid sequence of CD20CD3DVD-Ig Protein Protein SequenceSequence region Identifier 12345678901234567890 DVD HEAVY SEQ ID NO.: 97QVQLRQPGAELVKPGASVKM VARIABLE SCKASGYTFTSYNMHWVKQT CD20CD3DVD-IgPGQGLEWIGAIYPGNGDTSY NQKFKGKATLTADKSSSTAY MQLSSLTSEDSAVYYCARSHYGSNYVDYFDYWGQGTTLTV SSAKTTAPSVYPLAPQVQLQ QSGAELARPGASVKMSCKASGYTFTRYTMHWVKQRPGQGL EWIGYINPSRGYTNYNQKFK DKATLTTDKSSSTAYMQLSSLTSEDSAVYYCARYYDDHYC LDYWGQGTTLTVSS 5F1 VH SEQ ID NO.: 98QVQLRQPGAELVKPGASVKM SCKASGYTFTSYNMHWVKQT PGQGLEWIGAIYPGNGDTSYNQKFKGKATLTADKSSSTAY MQLSSLTSEDSAVYYCARSH YGSNYVDYFDYWGQGTTLTV SS LINKERSEQ ID NO.: 99 AKTTAPSVYPLAP OKT3 VH SEQ ID NO.: 100QVQLQQSGAELARPGASVKM SCKASGYTFTRYTMHWVKQR PGQGLEWIGYINPSRGYTNYNQKFKDKATLTTDKSSSTAY MQLSSLTSEDSAVYYCARYY DDHYCLDYWGQGTTLTVSS CH SEQ IDNO.: 34 ASTKGPSVFPLAPSSKSTSG GTAALGCLVKDYFPEPVTVS WNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQT YICNVNHKPSNTKVDKKVEP KSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTP EVTCVVVDVSHEDPEVKFNW YVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGK EYKCKVSNKALPAPIEKTIS KAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDI AVEWESNGQPENNYKTTPPV LDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYT QKSLSLSPGK CD20CD3DVD-Ig SEQ ID NO.: 101QIVLSQSPAILSASPGEKVT LIGHT MTCRASSSLSFMHWYQQKPG VARIABLESSPKPWIYATSNLASGVPAR FSGSGSGTSYSLTISRVEAE DAATYFCHQWSSNPLTFGAGTKLELKRADAAPTVSIFPPQ IVLTQSPAIMSASPGEKVTM TCSASSSVSYMNWYQQKSGTSPKRWIYDTSKLASGVPAHF RGSGSGTSYSLTISGMEAED AATYYCQQWSSNPFTFGSGT KLEINR5F1 VL SEQ ID NO.: 102 QIVLSQSPAILSASPGEKVT MTCRASSSLSFMHWYQQKPGSSPKPWIYATSNLASGVPAR FSGSGSGTSYSLTISRVEAE DAATYFCHQWSSNPLTFGAG TKLELKRLINKER SEQ ID NO.: 103 ADAAPTVSIFPP OKT3 VL SEQ ID NO.: 104QIVLTQSPAIMSASPGEKVT MTCSASSSVSYMNWYQQKSG TSPKRWIYDTSKLASGVPAHFRGSGSGTSYSLTISGMEAE DAATYYCQQWSSNPFTFGSG TKLEINR CL SEQ ID NO.: 36TVAAPSVFIFPPSDEQLKSG TASVVCLLNNFYPREAKVQW KVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEK HKVYACEVTHQGLSSPVTKS FNRGEC

Example 5 Generation of mIL-1α/βDVD-Ig

To study key issues concerning pharmacokinetics, in vivo efficacy,tissue penetration, and immunogenicity of DVD-Ig molecules,mouse-anti-mouse IL-1α/β DVD-Ig molecules were constructed as describedbelow.

Example 5.1 Construction of mIL-1α/βDVD-Ig

Mouse-anti-mouse IL-1α/β DVD-Ig molecules were constructed using twomouse anti-mouse IL-1α/β mAbs (9H10 and 10G11) generated from IL-1αβdouble KO mice. Mouse anti-mouse IL-1α, and mouse anti-mouse IL-1β,monoclonal antibodies were generated by immunizing IL-1α/β double KOmice with mouse IL-1α, or mouse IL-1β, respectively. One mouseanti-mouse IL-1α (Clone 9H10), and one mouse anti-mouse IL-1β mAb (clone10G11), were selected and used to generate mIL-1α/β DVD-Ig molecules.Various linker sizes and different domain orientations were tested. Thefinal functional mIL-1α/β DVD-Ig molecules was constructed in aorientation of V(anti-mIL-1β)-linker-V(anti-mIL-1β)-murine constantregion (Cγ2a and Cκ). The cloning, expression, and purificationprocedures were similar to that of the hIL-1α/β DVD-Ig. The cloning ofmIL-1α/β DVD-Ig was carried out using similar overlapping PCR andhomologous recombination as described for hIL-1α/β DVD3-Ig. Thesequences of mIL-1α/β DVD-Ig are shown below in Table 26:

TABLE 26 Amino acid sequence of mIL-1α/β DVD-Ig Protein Protein SequenceSequence region Identifier 12345678901234567890 mIL- SEQ ID NO.: 105EVQLQQSGPELVKPGTSVKM 1α/β DVD-Ig SCKTSGYTFTSYVMHWVKQK HEAVYPGQGLEWIGYIIPYNDNTKY VARIABLE NEKFKGKATLTSDKSSSTAY MELSSLTSEDSAVYYCARRNEYYGSSFFDYWGQGTTLTVS SAKTTAPSVYPLAPQVILKE SGPGILQPSQTLSLTCSFSGFSLSTYGTAVNWIRQPSGKG LEWLAQIGSDDRKLYNPFLK SRITLSEDTSNSQVFLKITSVDTEDSATYYCANGVMEYWG LGTSVTVSS 10G11 VH SEQ ID NO.: 106EVQLQQSGPELVKPGTSVKM SCKTSGYTFTSYVMHWVKQK PGQGLEWIGYIIPYNDNTKYNEKFKGKATLTSDKSSSTAY MELSSLTSEDSAVYYCARRN EYYGSSFFDYWGQGTTLTVSS LINKERSEQ ID NO.: 99 AKTTAPSVYPLAP 9H10 VH SEQ ID NO.: 107QVILKESGPGILQPSQTLSL TCSFSGFSLSTYGTAVNWIR QPSGKGLEWLAQIGSDDRKLYNPFLKSRITLSEDTSNSQV FLKITSVDTEDSATYYCANG VMEYWGLGTSVTVSS CH SEQ ID NO.:108 AKTTAPSVYPLAPVCGDTTG SSVTLGCLVKGYFPEPVTLT WNSGSLSSGVHTFPAVLQSDLYTLSSSVTVTSSTWPSQSI TCNVAHPASSTKVDKKIEPR GPTIKPCPPCKCPAPNLLGGPSVFIFPPKIKDVLMISLSP IVTCVVVDVSEDDPDVQISW FVNNVEVHTAQTQTHREDYNSTLRVVSALPIQHQDWMSGK EFKCKVNNKDLPAPIERTIS KPKGSVRAPQVYVLPPPEEEMTKKQVTLTCMVTDFMPEDI YVEWTNNGKTELNYKNTEPV LDSDGSYFMYSKLRVEKKNWVERNSYSCSVVHEGLHNHHT TKSFSRTPGK mIL- SEQ ID NO.: 109DIQMTQSPASLSASVGETVT 1α/β DVD-Ig ITCRGSGILHNYLVWYQQKQ LIGHTGKSPQLLVYSAKILADGVPS VARIABLE RFSGSGSGTQYSLKINSLQP EDFGSYYCQHFWSTPFTFGSGTKLEIKRADAAPTVSIFPP SIVMTQTPKFLLVSAGDRVT ITCKASQSVNHDVAWYQQMPGQSPKLLIYFASNRYTGVPD RFTGSGYGTDFTFTISTVQA EDLAVYFCQQDYSSPYTFGG GTKLEIKR10G11 VL SEQ ID NO.: 110 DIQMTQSPASLSASVGETVT ITCRGSGILHNYLVWYQQKQGKSPQLLVYSAKILADGVPS RFSGSGSGTQYSLKINSLQP EDFGSYYCQHFWSTPFTFGS GTKLEIKRLINKER SEQ ID NO.: 111 ADAAPTVSIFPP 9H10 VL SEQ ID NO.: 112SIVMTQTPKFLLVSAGDRVT ITCKASQSVNHDVAWYQQMP GQSPKLLIYFASNRYTGVPDRFTGSGYGTDFTFTISTVQA EDLAVYFCQQDYSSPYTFGG GTKLEIKR CL SEQ ID NO.: 113ADAAPTVSIFPPSSEQLTSG GASVVCFLNNFYPKDINVKW KIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYER HNSYTCEATHKTSTSPIVKS FNRNEC

Murine mIL-1α/β DVD-Ig retained affinity/in vitro potency against bothIL-1α and IL-1β. Table 27 shows the characterization of mAbs 9H10(anti-mIL-1β), 10G11 (anti-mIL-1β), and mIL-1α/β DVD-Ig.

TABLE 27 Characterization of mDVD4-Ig Antigen K_(D) (M) IC₅₀ (M) 9H10mIL-1α 1.73E−10 2.00E−10 10G11 mIL-1β 2.30E−10 3.70E−10 mIL-1α/βDVD-IgmIL-1α 7.66E−10 2.00E−09 mIL-1β 6.94E−10 8.00E−10

Example 5.2 In Vivo Activity of mIL-1α/βDVD-Ig in Arthritis Model

The therapeutic effects of anti-IL-1 alpha, anti-IL-1 beta, combinedanti-IL-1-alpha/anti-IL-1beta, and murine anti-IL-1 alpha/beta DVD4-Ig,were evaluated in a collagen-induced arthritis mouse model well known inthe art. Briefly, male DBA-1 mice were immunized with bovine type IIcollagen in CFA at the base of the tail. The mice were boosted withZymosan intraperitoneally (i.p) at day 21. After disease onset at day24-27, mice were selected and divided into separate groups of 10 miceeach. The mean arthritis score of the control group, and anti-cytokinegroups was comparable at the start of treatment. To neutralize IL-1,mice were injected every other day with 1-3 mg/kg of anti-IL-1 alphamAb, anti-IL-1 beta mAb, combination of anti-IL-1-alpha/anti-IL-1 betamAbs, or murine anti-IL-1alpha/beta DVD4-Ig intraperitoneally. Mice werecarefully examined three times a week for the visual appearance ofarthritis in peripheral joints, and scores for disease activitydetermined.

Blockade of IL-1 in the therapeutic mode effectively reduced theseverity of arthritis, with anti-IL-1 beta showing greater efficacy (24%reduction in mean arthritis score compared to control group) thananti-IL-1-alpha (10% reduction). An additive effect was observed betweenanti-IL-1-alpha and anti-IL-1beta, with a 40% reduction in meanarthritis score in mice treated with both anti-IL-1alpha andanti-IL-1beta mAbs. Surprisingly, at the same dose level, the treatmentof mDVD-Ig exhibited 47% reduction in mean arthritis score,demonstrating the in vivo therapeutic efficacy of mDVD-Ig. Similarefficacy was also observed in the measurements of joint swelling in thisanimal model.

The present invention incorporates by reference in their entiretytechniques well known in the field of molecular biology and drugdelivery. These techniques include, but are not limited to, techniquesdescribed in the following publications:

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Although a number of embodiments and features have been described above,it will be understood by those skilled in the art that modifications andvariations of the described embodiments and features may be made withoutdeparting from the present disclosure or the invention as defined in theappended claims. Each of the publications mentioned herein isincorporated by reference.

1. A binding protein comprising first and second polypeptide chains,wherein said first polypeptide chain comprises VD1-(X1)n-VD2-C-(X2)n,wherein VD1 is a first heavy chain variable domain, VD2 is a secondheavy chain variable domain, C is a constant domain, X1 is a linker withthe proviso that it is not a constant domain, and X2 is an Fc region;and said second polypeptide chain comprises VD1-(X1)n-VD2-C-(X2)n,wherein VD1 is a first light chain variable domain, VD2 is a secondlight chain variable domain, C is a constant domain, X1 is a linker withthe proviso that it is not a constant domain, and X2 does not comprisean Fc region; wherein n is 0 or 1; and wherein said two polypeptidechains of said binding protein form two functional binding sites.
 2. Thebinding protein according to claim 1, wherein said binding protein iscapable of binding one or more targets.
 3. The binding protein accordingto claim 2, wherein the target is selected from the group consisting ofABCF1; ACVR1; ACVR1B; ACVR2; ACVR2B; ACVRL1; ADORA2A; Aggrecan; AGR2;AICDA; AIF1; AIG1; AKAP1; AKAP2; AMH; AMHR2; ANGPT1; ANGPT2; ANGPTL3;ANGPTL4; ANPEP; APC; APOC1; AR; AZGP1 (zinc-a-glycoprotein); B7.1; B7.2;BAD; BAFF; BAG1; BAI1; BCL2; BCL6; BDNF; BLNK; BLR1 (MDR15); BlyS; BMP1;BMP2; BMP3B (GDF10); BMP4; BMP6; BMP8; BMPR1A; BMPR1B; BMPR2; BPAG1(plectin); BRCA1; C19orf10 (IL27w); C3; C4A; C5; C5R1; CANT1; CASP1;CASP4; CAV1; CCBP2 (D6/JAB61); CCL1 (I-309); CCL11 (eotaxin); CCL13(MCP-4); CCL15 (MIP-1d); CCL16 (HCC-4); CCL17 (TARC); CCL18 (PARC);CCL19 (MIP-3b); CCL2 (MCP-1); MCAF; CCL20 (MIP-3a); CCL21(MIP-2); SLC;exodus-2; CCL22 (MDC/STC-1); CCL23 (MPIF-1); CCL24 (MPIF-2/eotaxin-2);CCL25 (TECK); CCL26 (eotaxin-3); CCL27 (CTACK/ILC); CCL28; CCL3(MIP-1a);CCL4 (MIP-1b); CCL5 (RANTES); CCL7 (MCP-3); CCL8 (mcp-2); CCNA1; CCNA2;CCND1; CCNE1; CCNE2; CCR1(CKR1/HM145); CCR2 (mcp-1RB/RA); CCR3(CKR3/CMKBR3); CCR4; CCR5 (CMKBR5/ChemR13); CCR6(CMKBR6/CKR-L3/STRL22/DRY6); CCR7 (CKR7/EBI1); CCR8(CMKBR8/TER1/CKR-L1); CCR9 (GPR-9-6); CCRL1 (VSHK1); CCRL2 (L-CCR);CD164; CD19; CD1C; CD20; CD200; CD-22; CD24; CD28; CD3; CD37; CD38;CD3E; CD3G; CD3Z; CD4; CD40; CD40L; CD44; CD45RB; CD52; CD69; CD72;CD74; CD79A; CD79B; CD8; CD80; CD81; CD83; CD86; CDH1 (E-cadherin);CDH10; CDH12; CDH13; CDH18; CDH19; CDH20; CDH5; CDH7; CDH8; CDH9; CDK2;CDK3; CDK4; CDK5; CDK6; CDK7; CDK9; CDKN1A (p21Wap1/Cip1); CDKN1B(p27Kip1); CDKN1C; CDKN2A (p16INK4a); CDKN2B; CDKN2C; CDKN3; CEBPB;CER1; CHGA; CHGB; Chitinase; CHST10; CKLFSF2; CKLFSF3; CKLFSF4; CKLFSF5;CKLFSF6; CKLFSF7; CKLFSF8; CLDN3; CLDN7 (claudin-7); CLN3; CLU(clusterin); CMKLR1; CMKOR1 (RDC1); CNR1; COL18A1; COL1A1; COL4A3;COL6A1; CR2; CRP; CSF1 (M-CSF); CSF2 (GM-CSF); CSF3 (GCSF); CTLA4;CTNNB1 (b-catenin); CTSB (cathepsin B); CX3CL1(SCYD1); CX3CR1 (V28);CXCL1 (GRO1); CXCL10 (IP-10); CXCL11 (I-TAC/IP-9); CXCL12 (SDF1);CXCL13; CXCL14; CXCL16; CXCL2 (GRO2); CXCL3 (GRO3); CXCL5 (ENA-78/LIX);CXCL6 (GCP-2); CXCL9 (MIG); CXCR3 (GPR9/CKR-L2); CXCR4; CXCR6 (TYMSTR/STRL33/Bonzo); CYB5; CYC1; CYSLTR1; DAB2IP; DES; DKFZp451J0118; DNCL1;DPP4; E2F1; ECGF1; EDG1; EFNA1; EFNA3; EFNB2; EGF; EGFR; ELAC2; ENG;ENO1; ENO2; ENO3; EPHB4; EPO; ERBB2 (Her-2); EREG; ERK8; ESR1; ESR2; F3(TF); FADD; FasL; FASN; FCER1A; FCER2; FCGR3A; FGF; FGF1 (aFGF); FGF10;FGF11; FGF12; FGF12B; FGF13; FGF14; FGF16; FGF17; FGF18; FGF19; FGF2(bFGF); FGF20; FGF21; FGF22; FGF23; FGF3 (int-2); FGF4(HST); FGF5; FGF6(HST-2); FGF7 (KGF); FGF8; FGF9; FGFR3; FIGF (VEGFD); FIL1(EPSILON);FIL1 (ZETA); FLJ12584; FLJ25530; FLRT1 (fibronectin); FLT1; FOS; FOSL1(FRA-1); FY (DARC); GABRP (GABAa); GAGEB1; GAGEC1; GALNAC4S-6ST; GATA3;GDF5; GFI1; GGT1; GM-CSF; GNAS1; GNRH1; GPR2 (CCR10); GPR31; GPR44;GPR81 (FKSG80); GRCC10 (C10); GRP; GSN (Gelsolin); GSTP1; HAVCR2; HDAC4;HDAC5; HDAC7A; HDAC9; HGF; HIF1A; HIP1; histamine and histaminereceptors; HLA-A; HLA-DRA; HM74; HMOX1; HUMCYT2A; ICEBERG; ICOSL; ID2;IFN-a; IFNA1; IFNA2; IFNA4; IFNA5; IFNA6; IFNA7; IFNB1; IFNgamma; IFNW1;IGBP1; IGF1; IGF1R; IGF2; IGFBP2; IGFBP3; IGFBP6; IL-1; IL10; IL10RA;IL10RB; IL11; IL11RA; IL-12; IL12A; IL12B; IL12RB1; IL12RB2; IL13;IL13RA1; IL13RA2; IL14; IL15; IL15RA; IL16; IL17; IL17B; IL17C; IL17R;IL18; IL18BP; IL18R1; IL18RAP; IL19; IL1A; IL1B; ID F10; ID F5; IL1F6;IL1F7; IL1F8; IL1F9; IL1HY1; IL1R1; IL1R2; IL1RAP; IL1RAPL1; IL1RAPL2;IL1RL1; IL1RL2IL1RN; IL2; IL20; IL20RA; IL21R; IL22; IL22R; IL22RA2;IL23; IL24; IL25; IL26; IL27; IL28A; IL28B; IL29; IL2RA; IL2RB; IL2RG;IL3; IL30; IL3RA; IL4; IL4R; IL5; IL5RA; IL6; IL6R; IL6ST (glycoprotein130); IL7; IL7R; IL8; IL8RA; IL8RB; IL8RB; IL9; IL9R; ILK; INHA; INHBA;INSL3; INSL4; IRAK1; IRAK2; ITGA1; ITGA2; ITGA3; ITGA6 (a6 integrin);ITGAV; ITGB3; ITGB4 (b 4 integrin); JAG1; JAK1; JAK3; JUN; K6HF; KAI1;KDR; KITLG; KLF5 (GC Box BP); KLF6; KLK10; KLK12; KLK13; KLK14; KLK15;KLK3; KLK4; KLK5; KLK6; KLK9; KRT1; KRT19(Keratin 19); KRT2A; KRTHB6(hair-specific type II keratin); LAMA5; LEP (leptin); Lingo-p75;Lingo-Troy; LPS; LTA (TNF-b); LTB; LTB4R (GPR16); LTB4R2; LTBR;MACMARCKS; MAG or Omgp ; MAP2K7 (c-Jun); MDK; MIB1; midkine; MIF; MIP-2;MK167 (Ki-67); MMP2; MMP9; MS4A1; MSMB; MT3 (metallothionectin-III);MTSS1; MUC1 (mucin); MYC; MYD88; NCK2; neurocan; NFKB1; NFKB2; NGFB(NGF); NGFR; NgR-Lingo; NgR-Nogo66 (Nogo); NgR-p75; NgR-Troy; NME1(NM23A); NOX5; NPPB; NR0B1; NR0B2; NR1D1; NR1D2; NR1H2; NR1H3; NR1H4;NR1I2; NR1I3; NR2C1; NR2C2; NR2E1; NR2E3; NR2F1; NR2F2; NR2F6; NR3C1;NR3C2; NR4A1; NR4A2; NR4A3; NR5A1; NR5A2; NR6A1; NRP1; NRP2; NT5E; NTN4;ODZ1; OPRD1; P2RX7; PAP; PART1; PATE; PAWR; PCA3; PCNA; PDGFA; PDGFB;PECAM1; PF4 (CXCL4); PGF; PGR; phosphacan; PIAS2; PIK3CG; PLAU (uPA);PLG; PLXDC1; PPBP (CXCL7); PPID; PR1; PRKCQ; PRKD1; PRL; PROC; PROK2;PSAP; PSCA; PTAFR; PTEN; PTGS2 (COX-2); PTN; RAC2 (p21Rac2); RARB; RGS1;RGS13; RGS3; RNF110 (ZNF144); ROBO2; S100A2; SCGB1D2 (lipophilin B);SCGB2A1(mammaglobin 2); SCGB2A2 (mammaglobin 1); SCYE1 (endothelialMonocyte-activating cytokine); SDF2; SERPINA1; SERPINA3; SERPINB5(maspin); SERPINE1(PAI-1); SERPINF1; SHBG; SLA2; SLC2A2; SLC33A1;SLC43A1; SLIT2; SPP1; SPRR1B (Spr1); ST6GAL1; STAB1; STAT6; STEAP;STEAP2; TB4R2; TBX21; TCP10; TDGF1; TEK; TGFA; TGFB1; TGFB1I1; TGFB2;TGFB3; TGFBI; TGFBR1; TGFBR2; TGFBR3; TH1L; THBS1 (thrombospondin-1);THBS2; THBS4; THPO; TIE (Tie-1); TIMP3; tissue factor; TLR10; TLR2;TLR3; TLR4; TLR5; TLR6; TLR7; TLR8; TLR9; TNF; TNF-a; TNFAIP2 (B94);TNFAIP3; TNFRSF11A; TNFRSF1A; TNFRSF1B; TNFRSF21; TNFRSF5; TNFRSF6(Fas); TNFRSF7; TNFRSF8; TNFRSF9; TNFSF10(TRAIL); TNFSF11 (TRANCE);TNFSF12 (APO3L); TNFSF13 (April); TNFSF13B; TNFSF14 (HVEM-L); TNFSF15(VEGI); TNFSF18; TNFSF4 (OX40ligand); TNFSF5(CD40ligand); TNFSF6 (Fast);TNFSF7 (CD27ligand); TNFSF8 (CD30ligand); TNFSF9 (4-1BB ligand); TOLLIP;Toll-like receptors; TOP2A (topoisomerase Iia); TP53; TPM1; TPM2; TRADD;TRAF1; TRAF2; TRAF3; TRAF4; TRAF5; TRAF6; TREM1; TREM2; TRPC6; TSLP;TWEAK; VEGF; VEGFB; VEGFC; versican; VHL C5; VLA-4; XCL1 (lymphotactin);XCL2 (SCM-1b); XCR1 (GPR5/CCXCR1); YY1; and ZFPM2.
 4. The bindingprotein according to claim 1, wherein said binding protein is capable ofbinding two targets, wherein the two targets are selected from the groupconsisting of CD138 and CD20; CD138 and CD40; CD20 and CD3; CD38 &CD138; CD38 and CD20; CD38 and CD40; CD40 and CD20; CD19 and CD20; CD-8and IL-6; PDL-1 and CTLA-4; CTLA-4 and BTNO2; CSPGs and RGM A; IGF1 andIGF2; IGF1/2 and Erb2B; IL-12 and IL-18; IL-12 and TWEAK; IL-13 andADAM8; IL-13 and CL25; IL-13 and IL-1beta ; IL-13and IL-25; IL-13andIL-4; IL-13and IL-5; IL-13 and IL-9; IL-13 and LHR agonist; IL-13 andMDC; IL-13 and MIF; IL-13 and PED2; IL-13 and SPRR2a; IL-13 and SPRR2b;IL-13 and TARC; IL-13 and TGF-β; IL-1α and IL-1β; MAG and RGM A; NgR andRGM A; NogoA and RGM A; OMGp and RGM A; RGM A and RGM B; Te38 and TNFα;TNFα and IL-12; TNFα and IL-12p40; TNFα and IL-13; TNFα and IL-15; TNFαand IL-17; TNFα and IL-18; TNFα and IL-1 beta; TNFα and IL-23; TNFα andMIF; TNFα and PEG2; TNFα and PGE4; TNFα and VEGF; and VEGFR and EGFR;TNFα and RANK ligand; TNFα and Blys; TNFα and GP130; TNFα and CD-22; andTNFα and CTLA-4.
 5. The binding protein according to claim 2, whereinthe binding protein is capable of modulating a biological function ofone or more targets.
 6. The binding protein according to claim 2,wherein the binding protein is capable of neutralizing one or moretargets.
 7. The binding protein according to claim 2, wherein the targetis selected from the group consisting of cytokine, chemokine, cellsurface protein, enzyme and receptor.
 8. The binding protein accordingto claim 7 wherein the cytokine is selected from the group consisting oflymphokines, monokines, and polypeptide hormones.
 9. The binding proteinaccording to claim 8, wherein said cytokines are IL-1α and IL-1α. 10.The binding protein according to claim 9, wherein the binding proteincomprises a DVD heavy chain amino acid sequence selected from the groupconsisting of SEQ ID NO. 33, SEQ ID NO. 37, SEQ ID NO. 41, SEQ ID NO.45, SEQ ID NO. 47, SEQ ID NO. 51, SEQ ID NO. 53, SEQ ID NO. 55, SEQ IDNO. 57, and SEQ ID NO. 59; and a DVD light chain amino acid sequenceselected from the group consisting of SEQ ID NO. 35, SEQ ID NO. 39, SEQID NO. 43, SEQ ID NO. 46, SEQ ID NO. 49, SEQ ID NO. 52, SEQ ID NO. 54,SEQ ID NO. 56, SEQ ID NO. 58, and SEQ ID NO.
 60. 11. The binding proteinaccording to claim 8, wherein said cytokines are TNF-α and IL-13. 12.The binding protein according to claim 8, wherein said cytokines areIL-12and IL-18.
 13. The binding protein according to claim 12, whereinthe binding protein comprises a DVD heavy chain amino acid sequenceselected from the group consisting of SEQ ID NO. 83, SEQ ID NO. 90, SEQID NO. 93, SEQ ID NO. 95, and SEQ ID NO. 114; and a DVD light chainamino acid sequence selected from the group consisting of SEQ ID NO. 86,SEQ ID NO. 91, SEQ ID NO. 94, SEQ ID NO. 96, and SEQ ID NO.
 116. 14. Thebinding protein according to claim 7 wherein the chemokine is selectedfrom the group consisting of CCR2, CCR5and CXCL-13.
 15. The bindingprotein according to claim 7 wherein the cell surface protein is anintegrin.
 16. The binding protein according to claim 7 wherein the cellsurface proteins are CD-20 and CD3.
 17. The binding protein according toclaim 16, wherein the binding protein comprises a DVD heavy chain aminoacid sequence that is SEQ ID NO. 97, and a DVD light chain amino acidsequence that is SEQ ID NO.
 101. 18. The binding protein according toclaim 7 wherein the enzyme is selected from the group consisting ofkinases and proteases.
 19. The binding protein according to claim 7wherein the receptor is selected from the group consisting of lymphokinereceptor, monokine receptor, and polypeptide hormone receptor.
 20. Thebinding protein according to claim 7, wherein said binding protein hasan on rate constant (Kon) to said one or more targets selected from thegroup consisting of: at least about 10²M⁻¹s⁻¹; at least about 10³M⁻¹s⁻¹;at least about 10⁴M⁻¹s⁻¹; at least about 10⁵M⁻¹s⁻¹; and at least about10⁶M⁻¹s⁻¹, as measured by surface plasmon resonance.
 21. The bindingprotein according to claim 7, wherein said binding protein has an offrate constant (Koff) to said one or more targets selected from the groupconsisting of: at most about 10⁻³s⁻¹; at most about 10⁻⁴s⁻¹; at mostabout 10⁻⁵s⁻¹; and at most about 10⁻⁶s⁻¹, as measured by surface plasmonresonance.
 22. The binding protein according to claim 7, wherein saidbinding protein has a dissociation constant (K_(D)) to said one or moretargets selected from the group consisting of: at most about 10⁻⁷ M; atmost about 10⁻⁸ M; at most about 10⁻⁹ M; at most about 10⁻¹⁰ M; at mostabout 10⁻¹¹ M; at most about 10⁻¹² M; and at most 10⁻¹³M.
 23. A bindingprotein conjugate comprising a binding protein described in claim 1,said binding protein conjugate further comprising an agent selected fromthe group consisting of: an immunoadhension molecule, an imaging agent,a therapeutic agent, and a cytotoxic agent.
 24. The binding proteinconjugate according to claim 23, wherein said agent is an imaging agentselected from the group consisting of a radiolabel, an enzyme, afluorescent label, a luminescent label, a bioluminescent label, amagnetic label, and biotin.
 25. The binding protein conjugate accordingto claim 24, wherein said imaging agent is a radiolabel selected fromthe group consisting of: ³H, ¹⁴C, ³⁵S_(,) ⁹⁰Y, ⁹⁹Tc, ¹¹¹In, ¹²⁵I, ₁₃₁I,¹⁷⁷Lu, ¹⁶⁶Ho, and ¹⁵³Sm.
 26. The binding protein conjugate according toclaim 24, wherein said agent is a therapeutic or cytotoxic agentselected from the group consisting of; an anti-metabolite, an alkylatingagent, an antibiotic, a growth factor, a cytokine, an anti-angiogenicagent, an anti-mitotic agent, an anthracycline, a toxin, and anapoptotic agent.
 27. A binding protein as described in claim 1, whereinsaid binding protein is produced according to a method comprisingculturing a host cell comprising a vector encoding said binding proteinin culture medium under conditions sufficient to produce said bindingprotein.
 28. A pharmaceutical composition comprising the binding proteindescribed in any one of claims 1, 3, and 23, and a pharmaceuticallyacceptable carrier.
 29. The pharmaceutical composition of claim 28further comprising at least one additional therapeutic agent.
 30. Thepharmaceutical composition of claim 29, wherein said additional agent isselected from the group consisting of: Therapeutic agent, imaging agent,cytotoxic agent, angiogenesis inhibitors; kinase inhibitors;co-stimulation molecule blockers; adhesion molecule blockers;anti-cytokine antibody or functional fragment thereof; methotrexate;cyclosporin; rapamycin; FK506; detectable label or reporter; a TNFantagonist; an antirheumatic; a muscle relaxant, a narcotic, anon-steroid anti-inflammatory drug (NSAID), an analgesic, an anesthetic,a sedative, a local anesthetic, a neuromuscular blocker, anantimicrobial, an antipsoriatic, a corticosteroid, an anabolic steroid,an erythropoietin, an immunization, an immunoglobulin, animmunosuppressive, a growth hormone, a hormone replacement drug, aradiopharmaceutical, an antidepressant, an antipsychotic, a stimulant,an asthma medication, a beta agonist, an inhaled steroid, an epinephrineor analog, a cytokine, and a cytokine antagonist.
 31. A binding proteincomprising first and second polypeptide chains, wherein: said firstpolypeptide chain comprises VD1-(X1)n-VD2-C-(X2)n, wherein VD1 is afirst variable domain, VD2 is a second variable domain, C is a constantdomain, X1 is a linker with the proviso that it is not a constantdomain, X2 is an Fc region, and n is 0 or 1; and said second polypeptidechain comprises VD1-(X1)n-VD2-C, wherein VD1 is a first variable domain,VD2 is a second variable domain, C is a constant domain, and X1 is alinker with the proviso that it is not a constant domain, and n is 0 or1; wherein said two polypeptide chains of said binding protein form twofunctional binding domains.
 32. The binding protein according to any oneof claims 1, 2-8, 12, 13, and 31, wherein the linker X1 is selected fromthe group consisting of AKTTPKLEEGEFSEAR (SEQ ID NO:118);AKTTPKLEEGEFSEARV (SEQ ID NO:119); AKTTPKLGG (SEQ ID NO:120); SAKTTPKLGG(SEQ ID NO:121); SAKTTP (SEQ ID NO:122); RADAAP (SEQ ID NO:123);RADAAPTVS (SEQ ID NO:124); RADAAAAGGPGS (SEQ ID NO:125); RADAAAA(G₄S)₄(SEQ ID NO:126); SAKTTPKLEEGEFSEARV (SEQ ID NO:127); ADAAP (SEQ IDNO:40); ADAAPTVSIFPP (SEQ ID NO:103); TVAAP (SEQ ID NO:44); TVAAPSVFIFPP(SEQ ID NO:50); QPKAAP (SEQ ID NO:88); QPKAAPSVTLFPP (SEQ ID NO:92);AKTTPP (SEQ ID NO:38); AKTTPPSVTPLAP (SEQ ID NO:128); akttap (SEQ IDNO:129); akttapsvyplap (SEQ ID NO:99); ASTKGP (SEQ ID NO:42);ASTKGPSVFPLAP (SEQ ID NO:48); GGGGSGGGGSGGGGS (SEQ ID NO:130);GENKVEYAPALMALS (SEQ ID NO:131); GPAKELTPLKEAKVS (SEQ ID NO:132); andGHEAAAVMQVQYPAS (SEQ ID NO:133).
 33. The binding protein according toclaim 1 or claim 31, wherein C in said first polypeptide chain is CH1,and wherein C in said second polypeptide chain is CL.
 34. The bindingprotein according to claim 1 or claim 31, wherein C in said firstpolypeptide chain is CL, and wherein C in said second polypeptide chainis CH1.
 35. The binding protein according to claim 31, wherein VD1 ofsaid first polypeptide chain is an immunoglobulin variable domain, VD2of said first polypeptide chain is an immunoglobulin variable domain,VD1 of said second polypeptide chain is an immunoglobulin variabledomain, and VD2 of said second polypeptide chain is an immunoglobulinvariable domain.
 36. The binding protein according to claim 31, whereinVD1 of said first polypeptide chain is a non-immunoglobulin variabledomain, VD2 of said first polypeptide chain is an immunoglobulinvariable domain, VD1 of said second polypeptide chain is anon-immunoglobulin variable domain, and VD2 of said second polypeptidechain is an immunoglobulin variable domain.
 37. The binding proteinaccording to claim 31, wherein VD1 of said first polypeptide chain is anon-immunoglobulin variable domain, VD2 of said first polypeptide chainis a non-immunoglobulin variable domain, VD1 of said second polypeptidechain is a non-immunoglobulin variable domain, and VD2 of said secondpolypeptide chain is a non-immunoglobulin variable domain.
 38. Thebinding protein according to claim 36 or claim 37, wherein VD1 of saidfirst polypeptide chain and VD1 of said second polypeptide chain form aligand binding domain of a receptor.
 39. The binding protein accordingto claim 37, wherein VD1 of said first polypeptide chain and VD1 of saidsecond polypeptide chain form a ligand binding domain of a receptor, andwherein VD2 of said first polypeptide chain and VD2 of said secondpolypeptide chain form a ligand binding domain of a receptor.
 40. Thebinding protein according to claim 1 or claim 31, wherein said bindingprotein is a crystallized binding protein.
 41. The crystallized bindingprotein according to claim 40, wherein said crystal is a carrier-freepharmaceutical controlled release crystal.
 42. The crystallized bindingprotein according to claim 40, wherein said binding protein has agreater half life in vivo than the soluble counterpart of said bindingprotein.
 43. The crystallized binding protein according to claim 40,wherein said binding protein retains biological activity.
 44. A methodof producing a binding protein, comprising expressing the bindingprotein of claim 1 or 31 in culture medium under conditions sufficientto produce the binding protein.
 45. The method of claim 44, wherein50-75% of the binding protein produced is a dual specific tetravalentbinding protein.
 46. The method of claim 44, wherein 75-90% of thebinding protein produced is a dual specific tetravalent binding protein.47. The method of claim 44, wherein 90-95% of the binding proteinproduced is a dual specific tetravalent binding protein.